L&H cells in lymphocyte-predominant Hodgkin's disease.
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B-Lymphocytes↗
Biomedical subjects
R Küppers
Publications and source records attributed to R Küppers.
Frequent occurrence of deletions and duplications during somatic hypermutation: implications for oncogene translocations and heavy chain disease.
Human naive and germinal center (GC) B cells were sorted by flow cytometry and rearranged VH region genes were amplified and sequenced from single cells. Whereas no deletions or insertions were found in naive B cells, approximately 4% of in-frame and >40% of out-of-frame rearrangements of GC B cells harbored deletions and/or insertions of variable length. The pattern of deletions/insertions and their restriction to mutated V genes strongly suggests that they result from somatic hypermutation. Deletions and insertions account for approximately 6% of somatic mutations introduced into rearranged VH region genes of GC B cells. These deletions/insertions seem to be the main cause for the generation of heavy chain disease proteins. Furthermore, it appears that several types of oncogene translocations (like c-myc translocations in Burkitt's lymphoma) occur as a byproduct of somatic hypermutation within the GC-and not during V(D)J recombination in the bone marrow as previously thought.
B-Lymphocytes↗
Amplification of TCRbeta gene rearrangements from micromanipulated single cells: T cells rosetting around Hodgkin and Reed-Sternberg cells in Hodgkin's disease are polyclonal.
Despite accounting for only a minor fraction of all cells in Hodgkin's lymphoma tissue, the Hodgkin and Reed-Sternberg (HRS) cells represent the malignant tumor cell clone in Hodgkin's disease (HD). By far the most abundant cell type in the tumor tissue are CD4+ T cells. Some of them intimately associate with HRS cells forming rosettes around them. This study addresses the question whether the rosetting phenomenon reflects a specific interaction between T and HRS cells by asking whether the rosettes are composed of T cells expressing a restricted TCR repertoire. Single rosetting T cells were micromanipulated from frozen sections of tumor tissue in two cases of nodular sclerosing HD and one case of lymphocyte predominant HD. TCR Vbeta gene rearrangements were amplified from these single cells by PCR. Of 83 potentially functional Vbeta gene rearrangements obtained altogether from the three cases, 81 were found to be clonally unrelated. Furthermore, they did not show signs of selection of the receptor chains for recognition of common epitopes: The usage of Vbeta and Jbeta gene segments as well as the distribution of complementarity-determining region (CDR) 3 lengths was similar to what was seen in a collection of 60 Vbeta gene rearrangements from blood of healthy donors and no recurrent CDR3 amino acid motifs were found. These data suggest that the HRS cells attract CD4+ T cells nonspecifically.
Adult↗
Human IgA- and IgM-secreting intestinal plasma cells carry heavily mutated VH region genes.
Single IgA- or IgM-secreting plasma cells were isolated from histological sections of human jejunum and terminal ileum, and Ig heavy chain variable (VH) region genes were amplified and sequenced. Taken together, 62 of 63 cells analyzed harbored somatically mutated VH region genes, indicating that the vast majority of both IgA- and IgM-secreting intestinal plasma cells derive from germinal center B cells. On average, rearranged VH genes of IgA- and IgM-secreting plasma cells showed a mutation frequency of 9.0 % and 8.5 %, respectively, which exceeds the level of somatic mutation of V region genes carried by human memory B cells. Moreover, we detected deletions or insertions in the complementarity-determining regions of 5 of the 58 functional VH region genes analyzed, suggesting that these alterations may contribute to the diversification of the human antibody repertoire in the course of an immune reaction.
Genes, Immunoglobulin↗
Detection of identical Hodgkin-Reed Sternberg cell specific immunoglobulin gene rearrangements in a patient with Hodgkin's disease of mixed cellularity subtype at primary diagnosis and in relapse two and a half years later.
BACKGROUND: The malignant nature of Hodgkin-Reed Sternberg (H-RS) cells has been questioned due to their scarcity in lymphoma tissues. Recently, using micromanipulation of H-RS cells and single cell PCR evidence was obtained that H-RS cells represent a clonal B-cell population. In these studies H-RS cells were isolated from each one lymph node for a given case. In classical Hodgkin's disease (HD) it thus could not be ruled out that H-RS cell clonality reflected a locally restricted clonal proliferation. We analysed biopsy specimens from a patient suffering from HD for the presence of clonally related H-RS cells at primary diagnosis and during relapse of the disease. MATERIALS AND METHODS: In 1994 the H-RS cell line L1236 was generated from the peripheral blood of a patient suffering from a disseminating relapse of HD of mixed cellularity subtype. The patient had relapsed despite intensive treatment including high dose chemotherapy and autologous bone marrow transplantation. The clonal identity of this cell line with H-RS cells in situ was proven by amplifying identical Ig gene rearrangements of the cell line as well as of single H-RS cells picked from the patients bone marrow. Primers covering the CDR3 region were chosen from the H-RS cell specific VH1 gene rearrangement to detect H-RS cells of the identical clone by amplifying the rearranged VH1 genes in tissue samples obtained during disseminating relapsing disease and at primary diagnosis of HD in 1991. RESULTS: The H-RS cell specific DNA sequence was detected in all affected tissues analysed including the cervical lymph node which has been exstirpated at primary diagnosis. CONCLUSION: This finding indicates the existence of a clonal H-RS cell population during the first manifestation of HD and persistence and dissemination of this clone despite aggressive treatment. Thus, in the described case the malignant nature of H-RS cells defined by dissemination and recurrence of the identical H-RS cell clone in relapsing disease is proven.
Adult↗
Clonality and germinal centre B-cell derivation of Hodgkin/Reed-Sternberg cells in Hodgkin's disease.
Molecular single-cell studies of Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin's disease (HD) have revealed the clonal nature of these peculiar tumour cells. HRS cells in classical HD as well as lymphocyte predominant (LP) HD originate from germinal center (GC) B cells in most cases, if not all. Whereas HRS cells in LP HD represent transformed antigen-selected GC B cells with evidence of ongoing immunoglobulin (Ig) V gene mutation, HRS cells in classical HD appear to often or always derive from GC B cells that have lost the capacity to express a functional antigen receptor. Using Ig gene rearrangements amplified from HRS cells as clonal markers for the tumour cells, it could be shown that the same HRS cell clone can disseminate in the patient and persist throughout the course of the disease. A common derivation of the tumour cells was recently demonstrated in two cases representing combinations of HD and non-Hodgkin's lymphoma. Finally, V-gene analysis showed that viable cells enriched by magnetic cell sorting from HD patients as HRS cells indeed represent the HRS-cell population of the patient.
Antigens, Neoplasm↗
Somatic hypermutation in normal and transformed human B cells.
In the human, most IgM+IgD+ as well as CD5+ peripheral blood B cells express unmutated V genes and thus can be assigned to a pre-germinal centre (GC) stage of development. The memory B-cell compartment generated in the GC reaction and characterized by cells bearing somatically mutated V-region genes consists not only of class-switched cells, but also of IgM-only B cells and perhaps a subset of IgM+IgD+B cells expressing the CD27 antigen. Comparison of the rearranged V-region genes of human B-cell lymphomas with those of the normal B-cell subsets allows the identification of the progenitor cells of these tumours in terms of their stage of maturation. On this basis, most B-cell non-Hodgkin lymphomas, and in addition Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin's disease (HD), are derived from B cells at a GC or post-GC stage of development. The mutation pattern indicates that the precursors of the tumour clones have been stringently selected for expression of a functional antigen receptor with one notable exception: HRS cells in classical (but not lymphocyte-predominant) HD appear to be derived from "crippled" GC B cells. Sequence analysis of rearranged V genes amplified from single tonsillar GC B cells revealed that the somatic hypermutation process introduces deletions and/or insertions into V-region genes more frequently than indicated by previous investigations. Presumably, this feature of the hypermutation mechanism is often responsible for the generation of heavy chain disease, and also several types of chromosomal translocations of oncogenes into immunoglobulin loci in human B-cell lymphomas.
Animals↗
The origin of Hodgkin and Reed/Sternberg cells in Hodgkin's disease.
One of the characteristic features of Hodgkin's disease (HD) is the presence of a small population of often bizarre-looking large mono- or multinucleated Hodgkin and Reed-Sternberg (HRS) cells within the affected tissue. Recent cytogenetic investigations, studies of Epstein-Barr virus (EBV) genomes present in HRS cells, and analyses of Ig gene rearrangements amplified from single, micromanipulated HRS cells show that these cells largely represent clonal populations. The finding of Ig gene rearrangements in HRS cells in most cases of HD identifies B cells as the precursors of HRS cells in most if not all cases. Furthermore, the presence and pattern of somatic mutations within the rearranged Ig genes show that HRS cells in classical (i.e. nodular sclerosis, mixed cellularity, and lymphocyte depletion HD) as well as lymphocyte predominant (LP) HD originate from germinal center (GC) B cells. Ongoing somatic mutation and evidence for selection link HRS cells from LP HD to a mutating, antigen-selected GC B cell. In classical HD, the finding of "crippling" mutations and lack of stringent selection for antigen receptor expression suggests that in this case HRS cells are derived from a compartment of GC B cells that were destined to die but escaped apoptosis by some transforming event. One candidate for the latter is EBV infection.
Animals↗
Molecular single-cell analysis reveals that CD5-positive peripheral blood B cells in healthy humans are characterized by rearranged Vkappa genes lacking somatic mutation.
B cells expressing the CD5 cell surface antigen are involved in certain B cell malignancies and autoimmune diseases. From studies in the mouse, it emerged that CD5+ B cells represent a separate lineage of B lymphocytes that, in contrast to conventional (CD5-) B cells, are not driven into T cell-dependent immune responses in which rearranged variable (V) region genes are diversified by somatic hypermutation. Against this background it came as a surprise that human disease-involved CD5-positive autoreactive B cells as well as B cell chronic lymphocytic leukemias can harbor somatically mutated V region genes. Recent V gene analyses on CD5+ B cells in healthy adults did not give rise to a clear picture about the fraction of somatically mutated among all CD5+ B cells. In this work we used a molecular single-cell analysis to determine reliably the frequency of mutated CD5+ B cells in healthy humans: single, kappa light chain-expressing CD5+ peripheral blood B cells were isolated by flow cytometry, and rearranged Vkappa genes were amplified by PCR. From one donor, CD5+CD19+ B cells were analyzed. Since CD5+ B cells were found among IgM+IgD+ and IgM+IgD- cells (but almost not among class-switched cells) from two other donors, individual cells corresponding to these IgM-expressing subsets were investigated separately. The sequence analysis of rearranged Vkappa genes revealed that most if not all CD5+ B cells in healthy humans carry unmutated V region genes. From one of the donors, a novel polymorphic Jkappa2 gene segment was identified. To explain the discrepancy between the frequent occurrence of disease-associated somatically mutated CD5+ B cells and the low incidence or absence of somatic mutation in normal CD5+ B cells, we speculate that CD5+ B cells usually do not participate in germinal center reactions, but if they occasionally do so, they may be at an increased risk to become involved in autoimmune diseases or B cell malignancies.
Adult↗
Hodgkin and Reed-Sternberg cells in lymphocyte predominant Hodgkin disease represent clonal populations of germinal center-derived tumor B cells.
Among the four subtypes of Hodgkin disease (HD), lymphocyte-predominant (LP) HD is now generally considered as a separate entity. The B cell nature of the typical Hodgkin and Reed-Sternberg (HRS) cells and their variants (L and H, lymphocytic and histiocytic cells) in LP HD has long been suspected, but the question of whether these cells represent a true tumor clone is unclear. We previously demonstrated clonal Ig gene rearrangements in one case of LP HD. In the present study, five cases of LP HD were analyzed by micromanipulation of single HRS cells from frozen tissue sections and DNA amplification of rearranged Ig heavy chain genes from those cells. Clonal V gene rearrangements harboring somatic mutations were detected in each case. In three cases ongoing somatic mutation was evident. This shows that HRS cells in LP HD are a clonal tumor population derived from germinal center B cells. The pattern of somatic mutation indicates that HRS cells in LP HD are selected for antibody expression. This, and the presence of ongoing mutation discriminates LP from classical HD.
Adolescent↗
Evidence for a large compartment of IgM-expressing memory B cells in humans.
The recent finding of somatically mutated mu heavy chain transcripts in human peripheral blood (PB) B lymphocytes suggests that T-dependent B-cell memory might not be restricted to class-switched cells. We provide here evidence that IgM-only PB B cells are likely to be the IgM-expressing counterpart of classical (IgM- IgD-) memory B cells in humans. As shown by molecular single cell analysis, most IgM-only cells carry mutated V region genes, like class-switched cells. Although both subsets represent populations of nonactivated, resting cells, they express higher levels of Ig mRNA than naive (IgM+ IgD+) B cells. IgM-only and class-switched cells are CD38- CD77-, and mostly CD23-, thus neither resembling germinal center nor naive B cells. Because many IgM-expressing B cells located in secondary lymphoid tissues resemble IgM-only PB B cells in terms of cell phenotype, we propose that the human lymphoid system contains a large compartment of IgM-expressing memory cells. Moreover, these cells seem to represent the nonmalignant counterparts of IgM-expressing tumor cells in sporadic Burkitt's lymphoma, MALT lymphoma, monocytoid B-cell lymphoma, and diffuse large-cell lymphoma that were found to harbor somatically mutated V genes.
B-Lymphocyte Subsets↗
Diffuse large cell lymphomas are derived from mature B cells carrying V region genes with a high load of somatic mutation and evidence of selection for antibody expression.
In the Revised European American Lymphoma (REAL) classification, several subtypes of high-grade lymphomas were combined in the entity diffuse large cell lymphoma (DLL). In the present study, a total of 19 cases of DLL (10 cases of centroblastic lymphoma, 5 cases of mediastinal B cell lymphoma, 2 cases of immunoblastic lymphoma, 1 case of T cell-rich B lymphoma and one case of large cell anaplastic lymphoma) were analyzed for somatically mutated immunoglobulin V region genes. Somatic mutations are acquired in the course of the germinal center (GC) reaction and are thus found in GC B cells and their descendants, i.e. memory B cells. The V gene sequences revealed that the tumor cells of all five subtypes of DLL harbored mutated V region genes and are thus derived from antigen-experienced (post) GC B cells. This indicates that from the point of view of the stage of development of the tumor precursor, the combination of those five subtypes to one entity, i.e. DLL, seems reasonable. In some cases, an unusually high frequency of somatic mutations was detected. This may indicate that DLL are derived from GC B cells, which, due to transforming events, stayed in the GC for prolonged periods of time, thereby accumulating a high load of somatic mutation. An analysis of the mutation pattern suggests that the tumor clone or its precursor were selected for antibody expression while acquiring somatic mutations. The latter observation discriminates DLL from classical Hodgkin's disease, where we recently also observed a high load of somatic mutation within rearranged V region genes, but a frequent occurrence of crippling mutations.
Adult↗
Normal and malignant B-cell development with special reference to Hodgkin's disease.
During their development, B lymphocytes are repeatedly selected for the expression of an appropriate surface receptor: the pre-B-cell receptor at the pre-B-cell stage and surface immunoglobulin (Ig) at the transition from a pre-B cell to a mature B cell. Furthermore, stringent selection for B cells expressing high affinity antibodies operates when antigen-activated B cells proliferate within germinal centers (GC). Here, somatic point mutations are introduced into rearranged V region genes at a high rate, and B cells acquiring favorable mutations are selected to differentiate into memory B cells or plasma cells. In the frame of this developmental scheme, extending a recent analysis, we investigated 10 primary cases of Hodgkin's disease (HD) for B-lineage origin and clonality [1]. Single Hodgkin and Reed-Sternberg (H-RS) cells were micromanipulated from frozen tissue sections and analyzed by PCR for rearranged V genes. Clonal VH and/or V kappa/ V delta gene rearrangements were obtained from 9 of the cases. This shows that H-RS cells represent a clonal, B-lineage-derived population of tumor cells. Somatic mutations were found in all clonal VH gene rearrangements. Interestingly, mutations leading to stop codons in in-frame V gene rearrangements were detected in four cases. Since GC B cells acquiring such crippling mutations are usually efficiently eliminated within the GC, the finding of those mutations indicates that H-RS cells are derived from precursors within the GC that escaped apoptosis by a transforming event.
Animals↗
Ongoing somatic mutation in mantle cell lymphomas questioned.
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Humans↗
Molecular single cell studies of normal and transformed lymphocytes.
The polymerase chain reaction allows the characterization of RNA and DNA sequences from single cells. Methods were established to analyse single cells isolated from suspension or by multicolour flow cytometry. We established a method to isolate single immunostained cells from frozen tissue sections and to analyse those cells for immunoglobulin gene rearrangements. This method was first used to study B cell differentiation within human germinal centres. In another series of experiments, Hodgkin and Reed-Sternberg (HRS) cells from a total of 14 cases of HD were analysed for B lineage derivation and clonality. In 13 of the 14 cases, clonal V gene rearrangements were identified. This shows that HRS cells generally represent the outgrowth of a clonal population of B cells. The detection of somatic mutations in all VH gene rearrangements amplified from HRS cells and the nature of those mutations identifies a GC B cell as the HRS precursor.
Gene Rearrangement↗
Hodgkin and Reed-Sternberg cells in Hodgkin's disease represent the outgrowth of a dominant tumor clone derived from (crippled) germinal center B cells.
In Hodgkin's disease (HD), the Hodgkin and Reed-Sternberg (HRS) cells represent only a minute population in the diseased tissue. The investigation of lineage derivation and clonal origin of these cells has yielded conflicting results. We have analyzed HRS cells micromanipulated from infiltrated tissue sections of 10 primary HD patients for rearranged V genes, extending a previous study. Clonally related rearrangements were found in nine cases, indicating that HRS cells represent a dominant clone of B lineage-derived cells in at least a large fraction of cases of HD. Rearranged VH genes from HRS cells carried a high load of somatic mutation, indicating that HRS cells are derived from germinal center (GC) cells or their progeny. Stop codons in some in-frame V gene rearrangements suggest that the HRS cell precursors reside inside GCs, have acquired crippling mutations that prevent antigenic selection, but escape apoptosis through some transforming event.
Adolescent↗
Molecular single cell analysis demonstrates the derivation of a peripheral blood-derived cell line (L1236) from the Hodgkin/Reed-Sternberg cells of a Hodgkin's lymphoma patient.
A novel cell line, L1236, was established from the peripheral blood of a patient with Hodgkin's disease (HD). Two Ig VH and one Vkappa gene rearrangements were amplified by polymerase chain reaction (PCR) from the DNA of this cell line, demonstrating its derivation from the B-cell lineage. To test the cell line for its clonal relationship to the Hodgkin/Reed-Sternberg (H-RS) cells in the patient, single H-RS cells were micromanipulated from tissue sections of a tumor-infiltrated bone marrow specimen from that patient and analyzed by PCR for Ig gene rearrangements. The same rearrangements detected in the cell line were also found repeatedly in H-RS cells of the biopsy material. Thus, L1236 is the first cell line established from a case of HD for which a derivation from the H-RS cells of the patient could be demonstrated. Furthermore, the selective isolation of identical V gene rearrangements from multiple single H-RS cells demonstrates that these cells represented a clonal population in the patient.
Adult↗
Immunoglobulin V genes in Reed-Sternberg cells.
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Gene Rearrangement, B-Lymphocyte↗