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PubMed1998

Multiplex PCR for TCR delta rearrangements: a rapid and specific approach for the detection and identification of immature and mature rearrangements in ALL.

The preferential occurrence of immature T-cell receptor (TCR) delta rearrangements (i.e. incomplete Ddelta2-Ddelta3 and Vdelta2-Ddelta3) in B-cell precursor acute lymphoblastic leukaemia (BCP ALL) and of predominantly mature rearrangements (incomplete Ddelta2-Jdelta1, complete Vdelta1, Vdelta2, Vdelta3 to Jdelta1) in T-lineage ALL prompted us to establish two separate multiplex PCR systems for the identification of clonal TCRdelta rearrangements. PCR products of the expected size for the specific rearrangements were detectable from a dilution of 100-1000 clonal cells in 150000 polyclonal cells. Both multiplex PCR systems were used to analyse samples from 86 childhood BCPALLs and 30 T-lineage ALLs. The results of the multiplex PCRs were controlled by standard PCR analyses for the individual rearrangements and Southern blots, which were identical. Only immature TCRdelta rearrangements were detected in BCP ALL (59%), whereas no rearrangement was found in the remaining BCP leukaemias, thus confirming the exclusive presence of immature TCRdelta rearrangements in B-lineage cells. 50% of the T-lineage ALLs contained mature rearrangements, but no immature rearrangements were found. These two multiplex PCR techniques appear to be reliable and fast aids in the analysis of clonal TCRdelta rearrangements in ALL.

Blotting, Southern↗
PubMed2002

Assessment of RET/PTC oncogene activation and clonality in thyroid nodules with incomplete morphological evidence of papillary carcinoma: a search for the early precursors of papillary cancer.

Noninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer are difficult to diagnose and we do not know if they represent papillary carcinoma precursor lesions. Forty-six such nodules were analyzed for RET activation by immunohistochemistry and, in selected cases, by reverse transcriptase-polymerase chain reaction performed on RNA extracted after laser capture microdissection (LCM) of the tumor foci with and without papillary carcinoma features and positive RET immunoreactivity. RET immunoreactivity was identified, at least focally, in 30 of 46 (65.2%) of the nodules where it closely paralleled the morphological changes. Enough RNA was obtained after LCM in seven samples. RET/PTC1 or RET/PTC3 were detected in microscopic foci with papillary carcinoma features in most of the thyroid nodules (five of seven cases). No RET/PTC1 or RET/PTC3 rearrangements were detected in areas of the same tumors that lacked the cytological alterations. Analysis of clonality in the same nodules selected for LCM demonstrated that two were monoclonal and six were polyclonal. We conclude that RET activation closely parallels the morphological changes, that it is restricted to those areas of the tumor with the cytological alterations and that it is detectable in both mono- and polyclonal tumors. Although the finding of microscopic foci indicative of papillary carcinoma in a hyperplastic or adenomatous nodule does not justify the interpretation of the entire lesion as papillary carcinoma, it is possible that such foci may precede the development of invasive papillary cancer.

3T3 Cells↗
PubMed1982

A reproducible colonial morphology formed by individual pluripotent embryonal carcinoma cells in culture and selection of variants.

Embryonal carcinoma cells are stem cells equivalent to those of the early embryo which can be grown in vitro and which under certain conditions can differentiate into many cell types. Events in this differentiation process are numerous and complex, thus a system for the analysis of clonal differentiation is essential. In this paper I report that individual pluripotent embryonal carcinoma cells can each give rise to colonies, in the absence of a feeder layer but in the presence of beta-mercaptoethanol, that show a distinctive and reproducible gross morphology. Embryonal carcinoma cell lines can be derived from the stem cells in these colonies. Furthermore, variant cell lines can be derived from those colonies showing an altered gross morphology. These lines when cloned as above give rise to colonies showing a gross colonial morphology different to that of wild-type. These variant lines have been shown to be embryonal carcinoma cell lines. These findings indicate that genetic and cell lineage analysis of embryonal carcinoma cell differentiation might be possible.

Animals↗
PubMed1990

[Clonality and stem cell defects in the molecular pathology of chronic myeloproliferative disorders].

The chronic myeloproliferative disorders (CMD) are characterized by sustained or progressive proliferations of bone marrow cells, affecting one or more lineages in varying combinations. They are classified in 4 subgroups but transitional forms and transformations among the different entities are common. Analysing RFLPs and methylation patterns of X-chromosomal genes we could show, that in each entity granulocytes as well as bone marrow cells are of monoclonal origin. These findings support the view that all forms of CMD have in common that they arise from a multipotent hematopoietic stem cell. CML can be differentiated from all other forms of CMD by the Philadelphia-chromosome which on the molecular level has been revealed as bcr-abl gene junction. We investigated 258 cases of CMD for rearrangement of the bcr gene and found that it selectively occurred in CML. The methods applied can be of diagnostic value in differentiating reactive from neoplastic proliferations by analysis of clonality, and in differentiation of CML from other types of CMD by detection of the bcr-rearrangement.

Bone Marrow↗
PubMed2001

Juxtaposition of an active promoter to vsp genes via site-specific DNA inversions generates antigenic variation in Mycoplasma bovis.

Mycoplasma bovis, the most important etiological agent of bovine mycoplasmosis, undergoes extensive antigenic variation of major and highly immunogenic surface lipoprotein antigens (Vsps). A family of 13 related but divergent vsp genes, which occur as single chromosomal copies, was recently found in the chromosome of M. bovis. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal isolates exhibiting phase transitions of VspA or of VspC (i.e., ON-->OFF-->ON) has shown that DNA inversions occur during Vsp phase variation. The upstream region of each vsp gene contains two sequence cassettes. The first (cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhibits 98% homology among all vsp genes, while the second (cassette no. 2), upstream of cassette no. 1, ranges in size from 50 to 180 bp and is more divergent. Examination of the ends of the inverted fragments during VspA or VspC phase variation revealed that in both cases, a change in the organization of vsp upstream cassettes involving three vsp genes had occurred. Primer extension and Northern blot analysis have shown that a specific cassette no. 2, designated A(2), is an active promoter and that juxtaposition of this regulatory element to a silent vsp gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that phase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present within the conserved cassette no. 1. A model for the control of Vsp phase variation is proposed.

Antigenic Variation↗
PubMed2026

Multiomic study of cutaneous T-cell lymphoma reveals single-cell clonal evolution in progression and therapy resistance.

Cutaneous T-cell lymphoma (CTCL) remains a challenging disease due to its significant heterogeneity, therapy resistance, and relentless progression. Multiomics technologies offer the potential to provide uniquely precise views of disease progression and response to therapy. Here, we present a comprehensive multiomics view of CTCL clonal evolution, incorporating exome, whole-genome, epigenome, bulk, single-cell T-cell receptor, and single-cell RNA sequencing of 99 clinically annotated serial skin, peripheral blood, and lymph node samples from 34 patients with CTCL. We leveraged this extensive data set to define the molecular underpinnings of CTCL progression in individual patients at single-cell resolution with the goal of identifying clinically useful biomarkers and therapeutic targets. Our studies identified recurrent progression-associated clonal genomic alterations; we highlight mutation of CCR4, phosphoinositide 3-kinase inhibitor signaling, and programmed cell death protein 1 (PD-1) checkpoint pathways as evasion tactics deployed by malignant T cells. We identified a gain-of-function mutation in STAT3 (D661Y) and demonstrated, using cleavage under targets and release using nuclease (CUT&RUN) and RNA sequencing, that it enhances binding to and transcription of genes in Rho GTPase pathways. With our previous work implicating this pathway in histone deacetylase inhibitor-resistant CTCL, these data provide further support for a previously unrecognized role for Rho GTPase pathway dysregulation in CTCL progression. Recurrent progression-associated mutations were common in the epigenetic modifier EZH2, suggesting that EZH2 inhibition may benefit patients with CTCL. Our findings support an approach in which genomic analysis is widely used for improved disease monitoring, biomarker-informed clinical trial design, and genome-guided therapeutic decision-making. Moreover, these molecular changes present new opportunities for therapeutic targeting in this challenging and incurable cancer.

Multiomics↗
PubMed2001

In vitro selection and characterization of Bcl-X(L)-binding proteins from a mix of tissue-specific mRNA display libraries.

The covalent coupling of an mRNA to the protein that it encodes (mRNA display) provides a powerful tool for analysis of protein function in the post-genomic era. This coupling allows the selective enrichment of individual members from libraries of displayed proteins and the subsequent regeneration of an enriched library using the RNA moiety. Tissue-specific libraries from poly(A)(+) mRNA were prepared by priming first and second strand cDNA synthesis with oligonucleotides containing nine random 3' nucleotides, the fixed regions of which encoded the requisite sequences for formation of mRNA display constructs and a library-specific sequence tag. Starting with a pool of uniquely tagged libraries from different tissues, an iterative selection was performed for binding partners of the anti-apoptotic protein Bcl-X(L). After four rounds of selection, the pool was deconvoluted by polymerase chain reaction amplification with library-specific primers. Subsequent clonal sequence analysis revealed the selection of three members of the Bcl-2 family known to bind to Bcl-X(L). In addition, several proteins not previously demonstrated to interact with Bcl-X(L) were identified. The relative binding affinities of individual selected peptides were determined, as was their susceptibility to competition with a BH3 domain peptide. Based on these data, a putative BH3 domain was identified in most peptides.

Amino Acid Sequence↗
PubMed2000

Clinical relevance of immunoglobulin mutation analysis.

The precise diagnosis of malignant B-cell lymphoproliferations is essential for their appropriate clinical management. Histologic and immunophenotypical features alone are often insufficient in discriminating between various lymphoma entities and subtypes. The analysis of clonally rearranged immunoglobulin chain genes of the tumor cells was therefore suggested as an additional parameter. This article reviews current knowledge and describes which conclusions can be drawn for lymphoma diagnosis based on the type of immunoglobulin chain gene rearrangement, as well as on the number and distribution of somatic immunoglobulin mutations. Although there are striking correlations between the clinical outcome and the immunoglobulin mutations in some entities, many additional studies are required in order to draw final conclusions for most types of lymphoma.

B-Lymphocytes↗
PubMed2003

Analysis of T-cell receptor beta chain variable gene segment usage in healthy adult responders and nonresponders to recombinant hepatitis B vaccine.

One to 10 per cent of healthy adult individuals do not produce protective levels of anti-hepatitis B surface (HBs) antibodies, following a standard vaccination protocol. Lack of an HBs antigen (Ag)-specific T-cell repertoire is amongst the possible defects, which may lead to humoral unresponsiveness and is the main objective of this study. We analysed TcR BV (T-cell receptor beta chain variable) gene usage in T lymphocytes from nine healthy adult responders and six nonresponders to recombinant HB vaccine, before and after booster vaccination. CD4+ and CD8+ T-cell populations were isolated from peripheral blood mononuclear cells by magnetic beads, and the expression of TcR BV genes in each population was investigated by reverse transcription polymerase chain reaction and hybridization with specific probe. When the usage of each TcR BV gene within CD4+ and CD8+ T cells of the responders was compared with that of nonresponders, statistically significant difference (P < 0.01) was noted for BV5S2-3 gene family in CD4+ T cells of nonresponders. Furthermore, individual vaccinees were shown to overexpress several TcR BV genes. To characterize the T-cell repertoire and determine their clonal nature, analysis of CDR3 length polymorphism was performed. Our results show that T-cell response to HBsAg is generally oligoclonal and involves multiple BV families. Furthermore, overexpressed individual TcR BV genes and CDR3 length distributions in response to HBsAg are subject-dependent. In conclusion, our results are not in line with the notion that defective TcR repertoire may be an explanation for unresponsiveness to recombinant HBsAg vaccine.

Adult↗
PubMed1989

[Clonal evolution in tumor cell population].

Clonal evolution which characterizes malignant tumors is the consequence of two antagonizing forces acting on the tumor cell population, namely, forces of diversion and conversion. The former makes cells to diverge through genetic and epigenetic instabilities which are the built-in characteristics of malignant cells. Possible causes of genetic instability are discussed. These include mistakes in DNA synthesis by an error-prone DNA polymerase, the nucleotide pool distartion and the overreplication of replication origins, abnormal DNA repair, high rate recombination, by expression of fragile sites and possibly by expression of retrotransposons, frequent nondisjunction of chromosomes as a consequence of gene dosage inbalance, and abnormal DNA methylation. The second force makes the resulting tumor cell population with heterogenous phenotypes to converge through selection by host defence mechanisms, competition for nutrients and oxygen among tumor cells, to cell interactions within tumor and between surrounding normal tissues. Genetic tagging of tumor cells with pSV 2neo facilitates the analysis of clonal evolution which results from diversion and conversion of tumor cells. Selective growth and metastasis of a clone in a mouse sarcoma population was demonstrated. Generation of dominant clones as well as drug resistant clones in tumor can be studied with this method.

Animals↗
PubMed1987

Phenotypic and functional characterization of recombinant interleukin 2 (rIL 2)-induced activated killer cells: analysis at the population and clonal levels.

In these studies we investigated the phenotypic and functional characteristics of human rIL 2-activated killer cells (LAK). By FACS sorting we separated PBL into Leu-11- and Leu-11+ cell fractions and cultured them for 4 days in 100 U/ml rIL 2. Under these culture conditions, cells of the Leu-11+ fraction acquired a stronger LAK activity against fresh autologous or allogeneic melanoma cells as compared with Leu-11- cells or unfractionated PBL. To better characterize the cells responsible for this cytolytic activity, we directly cloned Leu-11+ and Leu-11- FACS-sorted cells in the presence of 1% PHA, irradiated spleen feeder cells, and rIL 2. From 6 to 10% of the Leu-11+ cells and from 42 to 66% of the Leu-11- cells plated gave rise to clonal progenies that were tested simultaneously for cytolytic activity against fresh melanoma cells and NK-sensitive K562 target cells in a 4-hr 51Cr-release assay. Most of the Leu-11+ microcultures lysed fresh melanoma target cells (35 out of 38 and 26 out of 34 in two separate experiments), whereas only a few clones derived from the Leu-11- cell fraction had this capability (four out of 45 and one out of 41). All the clones lysing fresh melanoma cells also efficiently killed K562 target cells, whereas other clones lysing only K562 could be found among Leu-11+ and Leu-11- clones. Nine clones expressing LAK activity were tested for their reactivity against a panel of different tumor target cells. All clones were able to lyse a broad panel of target cells including NK-sensitive and NK-resistant cultured or noncultured human tumor target cells, as well as mouse tumor cell lines. Surface marker analysis of 14 clones displaying LAK activity, all derived from Leu-11+ cells, showed that they were all T3 (CD3)-, whereas 10 out of 14 expressed the T11 (CD2) antigen and only four were weakly stained by an anti-T8 (CD8) mAb. All 14 clones expressed the T40 (CD7) T cell marker and DR and LFA-1 antigens. Cytolysis inhibition experiments performed on a rIL 2-activated Leu-11+ population and on two LAK cell clones, both expressing T11 antigen, showed that anti-LFA-1 but not anti-T11 mAb could inhibit cytolysis of freshly derived tumor target cells.

Antibodies, Monoclonal↗
PubMed1992

Is Bordetella pertussis clonal?

OBJECTIVE: To establish whether Bordetella pertussis is essentially clonal. DESIGN: Analysis of restriction fragments of XbaI digests of DNA from clinical and control isolates of B pertussis by pulse field gel electrophoresis. MATERIALS: 105 isolates of B pertussis: 67 clinical isolates from throughout the United Kingdom and 23 from Germany (collected during the previous 18 months); vaccine strains 2991 and 3700; and 13 control isolates from Manchester University's culture collection. MAIN OUTCOME MEASURES: Frequency of DNA types according to country of origin and classical serotyping. RESULTS: 17 DNA types were identified on the basis of the variation in 11 fragments, banding at 200-412 kilobases; 15 types were found in the clinical and control isolates from the United Kingdom and seven in those from Germany. There was no correlation with serotype. DNA type 1 was the commonest overall (22/105 strains, 22%), predominating in serotypes 1,2 and 1,2,3 and including the vaccine strains but not the isolates from Germany. CONCLUSIONS: Current infections due to B pertussis are not caused by a clonal pathogen as multiple strains are circulating in a given population at one time. There is also considerable epidemiological variation in the pathogen population between countries. These findings may have implications for the design of acellular vaccines.

Bordetella pertussis↗
PubMed1990

Chromosomal abnormality limited to T4 lymphocytes in a patient with T-cell chronic lymphocytic leukaemia.

Cytogenetic, immunologic and DNA studies were performed on a patient with the T-cell type of chronic lymphocytic leukaemia. Standard G-banding karyotype analysis revealed clonal chromosome abnormalities with the karyotype 42-44,XY,-11,-12,-13,-16,-21,-22,i(8q),inv(14) (q11q32), t(15;?)(p11;?), +4-6mar/43-44,X,t(Y;14)(p11;q11),-11,-12, -13,-21,-22,i(6p),i(8q),inv(14)(q11q32),t(15;?)(q11;?), +1-3mar. MAC (morphology, antibody, chromosomes) methodology, which allows the immunophenotyping of mitotic cells, showed that the chromosome abnormalities were restricted to CD4-positive helper/inducer T lymphocytes and that CD8 suppressor/cytotoxic T cells and B lymphocytes possessed a normal karyotype. The results also indicate that the proportion of abnormal metaphases and the overall mitotic activity after 3.5 days of stimulation in vitro were highest when PHA and TPA were used as mitogens. When the culture period in the presence of PHA + IL-2 was extended, the proportion of the abnormal cell population decreased in direct relation to the length of the culture period, ranging from 100% at 3.5 d to 0% at 31 d after stimulation. Southern blotting analysis revealed rearrangements of both the immunoglobulin heavy chain gene and the beta T-cell receptor gene.

Aged↗
PubMed1994

The determined state of white expression in the Drosophila eye is modified by zeste1 in the wzm family of mutants.

Analysis of the whitezeste mottled (wzm) mutant family suggests that the zeste gene product functions in establishing and stabilizing a transcriptionally active chromatin domain for white locus expression. The z1 mutation reduces expression of paired or proximate copies of white, while single or unpaired copies maintain wild-type levels of expression. The wzm mutation, caused by the insertion of the retrotransposon BEL into the 5' intron of white, alters the zeste-white interaction to produce a mottled eye phenotype in hemizygous z1 wzm males. We have determined the molecular structure of four wzm derivatives. wzl results from the insertion of an additional transposable element into the 5' regulatory region of white. wzvl is a deletion of sequences upstream of the white locus. Two others, whalo and wcres, result from the transposition of wzm plus the entire verticals-roughest region into heterochromatin near the tip of chromosome 3L. They variegate for roughest but not for white; rather, the z1 effect on wzm now causes white expression to become non-autonomous and non-clonal. The analysis of these five mutations shows that the neomorphic zeste1 product, in combination with structural changes imposed by transposons and intercalary heterochromatin, modifies the determination and stability of white expression. We propose that the normal zeste product functions as part of a complex that stimulates transcription by changing chromatin conformation to establish and maintain transcriptionally active domains. The unpairing of homologs is proposed to be one of the initial results of conformational change, providing an explanation for the role of zeste in transvection.

Alleles↗
PubMed2000

Genetic and epigenetic changes in human epithelial cells immortalized by telomerase.

Exogenous expression of hTERT, the catalytic component of telomerase, is sufficient for the immortalization of human fibroblasts but insufficient for the immortalization of human foreskin keratinocytes (HFKs) and human mammary epithelial cells (HMECs). These latter cell types can overcome senescence by coexpression of hTERT and human papillomavirus (HPV) E7 or by expression of hTERT and loss of p16(INK4a) expression, indicating that the retinoblastoma (Rb) pathway, along with a telomere maintenance pathway, plays a role in determining the life span of epithelial cells. In this study, we further characterize hTERT-immortalized HFKs and human adenoid epithelial cells (HAKs) for genotypic and phenotypic alterations that are associated with immortalization. Of five hTERT-immortalized HFK and HAK cell lines examined, four exhibited repression of p16(INK4a) expression by promoter methylation or specific large-scale deletion of chromosome 9p, the location of p16(INK4a). Interestingly, one cell line exhibited complete down-regulation of expression of p14(ARF), with only slight down-regulation of expression of p16(INK4a). Yet, all of the immortal cells lines exhibited hyperphosphorylated Rb. Cytogenetic analysis revealed clonal chromosome aberrations in three of the five cell lines. All of the cell lines retained a growth block response with the expression of mutant ras. When grown on organotypic raft cultures, however, the hTERT-immortalized cells exhibited a maturation delay on terminal differentiation. Our results indicate that immortalization of epithelial cells may require both activation of telomerase and other genetic and/or epigenetic alterations that abrogate normal differentiation.

Animals↗
PubMed2002

Acute lymphoblastic leukaemia: diagnosis and classification.

Acute lymphoblastic leukaemia (ALL) is a heterogeneous disease with distinct biological and prognostic groupings. Diagnosis relies on traditional cytomorphological and immunohistochemical evaluation of the leukaemic blasts. Subsequently, cytogenetic analysis identifies clonal numeric and/or structural chromosomal abnormalities that may be present, thus confirming the subtype classification and providing important prognostic information for treatment planning. The major chromosomal abnormalities in ALL are t(9;22)(q34;q11), t(12;21)(p13;q22), t(4;11)(q21;q23), t(1;19)(q23;p13), 8q24 translocations and hyperdiploidy. Generally, hyperdiploidy, occurring most frequently in paediatric cases, is associated with a good prognosis, while hypodiploidy confers a poor prognosis. Among structural chromosomal abnormalities, the t(9;22)(q34;q11) resulting in the BCR/ABL fusion protein, and rearrangements of the MLL gene, confer a poor prognosis in both children and adults, while t(12;21)(p13;q22), resulting in the TEL/AML1 fusion protein, and del (12p) confer a good prognosis. More recently, additional diagnostic and prognostic information has been gained from fluorescence in situ hybridization (FISH) and DNA microarray techniques.

Cell Lineage↗
PubMed1992

Expression of M-CSF and its receptor (C-FMS) during factor-independent cell line evolution from hematopoietic progenitor cells cocultivated with gamma irradiated marrow stromal cell lines.

Gamma irradiation of plateau-phase clonal bone marrow stromal cell lines produces factor-independent growth of cocultivated clonal interleukin-3/granulocyte-macrophage colony-stimulating factor-dependent hematopoietic progenitor cell lines. The process is associated with three biologic changes including: (i) adherence of hematopoietic cells to stromal cells forming 'cobblestone islands'; (ii) an intermediate stage [during which the cells show proliferation in suspension in the presence in leukemogenic stromal factor (LSF), a factor similar to macrophage colony-stimulating factor (M-CSF) released by irradiated stromal cells, and transient hematopoietic cell surface expression of MAC-1, and c-fms (M-CSF receptor)]; and (iii) a third stage of factor-independence. A monoclonal antibody to M-CSF receptor inhibited proliferation of intermediate stage but not all factor-independent cell subclones. In the present studies, a subclonal factor-independent malignant subline of FDC-P1JL26 derived by cocultivation with gamma-irradiated stromal cells as well as the parent clone and intermediate stage cells were shown to express significant levels of M-CSF polyA+ mRNA and M-CSF of at least two sizes (23 and 15 kDa) as detected by 35S-methionine labelling and immunoprecipitation with polyclonal anti-M-CSF antiserum. There was no significant difference in intracellular M-CSF protein size between cells at each of the three stages of biologic change. This M-CSF was not detected on the cell surface by fluorescence-activated cell sorting (FACS). In contrast, c-fms expression at the cell surface was detected by FACS analysis and c-fms polyA+ mRNA was only detected during the intermediate stage of induction of factor-independence. FDC-P1JL26 parent cells, the subclone stimulated by LSF, and the factor-independent subclone, showed little or no detectable autophosphorylation of the c-fms receptor at tyrosine. There was no detectable rearrangement of the M-CSF or c-fms genes by Southern analysis between clonal lines during the three stages. While we cannot rule out an autocrine mechanism or mutated c-fms receptor mechanism, the data also suggest that evolution of hemopoietic cell factor-independence during cocultivation with irradiated stromal cells may involve a mechanism distal to the c-fms receptor/M-CSF interaction.

Animals↗
PubMed1991

CD4+ T cell clones specific for the human p97 melanoma-associated antigen can eradicate pulmonary metastases from a murine tumor expressing the p97 antigen.

p97 is a human tumor-associated Ag present on most melanoma cells that represents a possible target for immunologic attack. To evaluate the capacity of T cells reactive with this protein to promote elimination of melanoma cells expressing p97, a murine model was developed by transfecting a C3H/HeN melanoma with the p97 cDNA, generating p97-specific CD4+ T cells by in vivo immunization of C3H/HeN mice with a vaccinia/p97 recombinant virus followed by in vitro cloning with soluble p97 protein, and determining whether these CD4+ T cells could mediate rejection of pulmonary metastases. Characterization of the T cell clones demonstrated the presence of both I-Ak and I-Ek-restricted clones, although the majority of clones recognized p97 in the context of I-Ek. Analysis of clonal specificity using truncated p97 proteins revealed that at least three epitopes were immunogenic, and further studies with overlapping 15-amino acid peptides from a region of the p97 molecule defined by these truncated proteins identified an immunodominant epitope responsible for the majority of the I-Ek response. The T cell clones were not capable of directly recognizing the p97-expressing melanoma cells but responded to the tumor if syngeneic APC were present to process the tumor-derived p97 Ag. The therapeutic efficacy of these CD4+ T cell clones was evaluated in an adoptive therapy model in which mice bearing metastatic pulmonary lesions were treated by i.v. administration of the p97-specific cells. Despite the inability of the CD4+ clones to directly respond to or lyse the tumor cells, the clones were effective in promoting tumor eradication. In vitro studies demonstrated that this may have reflected secretion of lymphokines that activated macrophages to lyse the tumor. The results suggest that noncytolytic p97-specific CD4+ T cell clones can be effective in therapy of pulmonary melanoma metastases. Moreover, if human T cells reactive with the p97 protein could be generated, the expression of this tumor-associated Ag in melanoma cells might be adequate for such T cells to mediate a therapeutic antitumor response.

Animals↗