Surface Immunoglobulin Light Chain Research Articles

Epstein-Barr virus-transformed human precursor B cell lines: altered growth phenotype of lines with germ-line or rearranged but nonexpressed heavy chain genes.

A series of lymphoblastoid cell lines (LCLs) have been established by in vitro infection of fetal bone marrow and fetal liver cells with Epstein-Barr virus (EBV). While most lines showed the usual mature B cell phenotype, a small proportion were cytoplasmic and surface immunoglobulin (Ig) heavy and light chain negative. Analysis of gene rearrangements indicated that the Ig- lines were either germ-line or nonproductively rearranged when probed for JH and were in germ-line configuration for C chi; no mu or chi mRNA could be detected in such cells. Precursor B cell lines were indistinguishable from their normal Ig+ counterparts in their expression of a wide variety of cell surface markers including "activation" antigens usually associated with the lymphoblastoid state; even the single LCL showing germ-line heavy and light chain genes expressed B lineage-specific cell surface antigens. However, the Ig- lines were distinct from their Ig+ counterparts in three important respects: (a) they grew much more slowly and achieved lower saturation densities, (b) they showed unusually high proportions (8-16%) of cells in EBV-productive cycle, and (c) they contained unusually high proportions (up to 40%) of cells expressing free joining (J) chain. These results suggest that precursor B cells differ in their response to the growth-transforming effects of EBV such that the virus-cell interaction in precursor B cell lines is inherently less stable than in conventional LCL. In particular there may be a greater movement of cells out of cycle and along the B cell maturation pathway. It is possible that such movement leads in individual cells either to virus replication or to a "sterile" plasmacytoid differentiation with J chain expression in the absence of Ig synthesis.

The human lymph node germinal center cell: characterization and isolation by using two-color flow cytometry.

The germinal center of lymphoid tissues is a critical microenvironmental site of B cell activation and differentiation in response to antigenic stimuli. However, characterization of germinal center cells (GCC) in tissue sections has proved technically difficult. Therefore, we have employed two-color flow cytometric analysis of suspended human tonsillar lymphocytes in order to define more precisely the immunologic features of GCC. These cells were identified in suspension by virtue of their specific surface binding of the lectin peanut agglutinin (PNA), confirmed by tissue immunoperoxidase studies. Phycoerythrin-labeled lectin was used in combination with a variety of fluorescein-labeled antibodies in order to identify subpopulations of tonsillar lymphocytes. The majority of PNA+ cells were B cells, and both PNA+ and PNA- B cells stained for surface immunoglobulin light chains. PNA+ cells lacked surface IgD, but included cells with surface IgG and IgM. Both PNA+ and PNA- cells stained for B1, B2, BA-1, Leu-12, Leu-14, CR-I, and HLA-DR antigens, whereas CALLA was present only on PNA+ cells. There were differences between PNA+ and PNA- cells in the relative expression of B1 and B2 antigens, possibly reflecting differences in B cell activation or maturation. A small proportion of T cells were PNA+, including both helper/inducer and suppressor/cytotoxic phenotypes. PNA+ cells included both small and large lymphoid cells, and almost all DNA synthetic activity was associated with the large PNA+ cells. PNA+ B cells isolated by cell sorting had morphologic features characteristic of GCC. Therefore, PNA+ cells in suspension appeared to represent GCC, and features of these cells that cannot be convincingly shown in tissue section studies were demonstrated by flow cytometry.

Surface Immunoglobulin Light Chain Expression in Pre‐B Cell Leukemiasa

Cytofluorographic analysis of surface immunoglobulin (sIg) light chain clonal excess (CE), defined as (%kappa+ - %lambda+)/(%kappa+ + %lambda+) cells per discrete level of fluorescence intensity, was carried out on mononuclear cells of 32 leukemic patients. Eight demonstrated sIg light chain CE, including four blastic chronic myeloid leukemias (BL-CML), three "null" acute lymphoblastic leukemias (ALL), and one leukemic lymphoblastic lymphoma. Six of the leukemias demonstrated a kappa CE and two had a lambda CE. Sorted kappa+ PB cells from a BL-CML patient were shown to have a diploid DNA stem line and to bear the "common" ALL antigen. To provide further support for our finding of the expression of sIg light chains in ALL, we studied the REH cell line, derived from a "common" ALL patient and found cytoplasmic mu heavy chain and surface Ig lambda CE. Nucleic acid blotting experiments on REH revealed that both kappa genes had been deleted and that lambda genes had been rearranged, as expected in B cells expressing lambda light chains. Moreover, REH cells contained mu and lambda RNA. When REH cells were treated with TPA the amount of mu chain RNA increased by approximately fivefold and the amount of lambda chain RNA increased by approximately twofold. The finding of sIg light chain in pre-B cell leukemias and in the REH cell line, suggests that these leukemic cells are further differentiated along the B-cell lineage than was previously believed.

Diffuse large cell lymphoma in a patient with hairy cell leukemia: immunoglobulin gene analysis reveals separate clonal origins

A 75-year-old man with hairy cell leukemia (HCL) was found to have an immunoblastic lymphoma of the small bowel. Immunologic and genotypic characterization of these neoplasms revealed both the HCL and the immunoblastic lymphoma to be of the B cell lineage. The HCL expressed the HCL-associated antigens detected by the monoclonal antibodies HC-1 and HC-2, whereas the immunoblastic lymphoma failed to react with these antibodies. In addition, surface immunoglobulin light chains could not be accurately determined for the hairy cells, whereas the immunoblastic lymphoma was shown to express only kappa immunoglobulin light chains. These immunophenotype differences were compatible with either the clonal evolution of the HCL into the immunoblastic lymphoma or a separate clonal origin for these two neoplasms. An analysis of tumor DNA by Southern blot hybridization revealed different heavy-chain and kappa light-chain gene rearrangements in these two malignancies. Thus the occurrence of the large cell lymphoma most likely represents the emergence of a second clonally unrelated B cell malignancy.

Diffuse Large Cell Lymphoma in a Patient With Hairy Cell Leukemia: Immunoglobulin Gene Analysis Reveals Separate Clonal Origins

A 75-year-old man with hairy cell leukemia (HCL) was found to have an immunoblastic lymphoma of the small bowel. Immunologic and genotypic characterization of these neoplasms revealed both the HCL and the immunoblastic lymphoma to be of the B cell lineage. The HCL expressed the HCL-associated antigens detected by the monoclonal antibodies HC-1 and HC-2, whereas the immunoblastic lymphoma failed to react with these antibodies. In addition, surface immunoglobulin light chains could not be accurately determined for the hairy cells, whereas the immunoblastic lymphoma was shown to express only ? immunoglobulin light chains. These immunophenotype differences were compatible with either the clonal evolution of the HCL into the immunoblastic lymphoma or a separate clonal origin for these two neoplasms. An analysis of tumor DNA by Southern blot hybridization revealed different heavy-chain and ? light-chain gene rearrangements in these two malignancies. Thus the occurrence of the large cell lymphoma most likely represents the emergence of a second clonally unrelated B cell malignancy.

Detection of clonal excess in lymphoproliferative disease by kappa/lambda analysis: correlation with immunoglobulin gene DNA rearrangement

Previous studies have suggested that analysis of the distribution of surface immunoglobulin light chain isotypes by flow cytometry provides evidence for monoclonality of B cell tumors and may detect populations of circulating tumor cells in patients with lymphoproliferative disease. We have used simultaneous flow cytometry and DNA restriction enzyme analysis on 58 samples of tissue and blood to determine whether lymphocyte populations detected by "kappa/lambda" analysis are indeed monoclonal. In greater than 90% of cases, abnormalities detected by flow cytometry correlated with monoclonal rearrangements of immunoglobulin genes as detected by Southern blot analysis. By analyzing tissue and blood from the same patients, we have also demonstrated that monoclonal circulating cells detected by flow cytometry reflect peripheral circulating tumor cells, since DNA from these cells shows the same immunoglobulin rearrangement as DNA from the original tumors in these patients. Although mixing studies suggested that DNA rearrangement studies were more sensitive than was flow cytometry in detecting minor populations of monoclonal lymphocytes, we found only one case in which this affected the diagnostic accuracy of the kappa/lambda analysis, with one notable exception, that of detection of a monoclonal proliferation of B cells that did not express surface immunoglobulin. The kappa/lambda test thus offers a powerful diagnostic tool in the evaluation of lymphoproliferative disease.

Detection of Clonal Excess in Lymphoproliferative Disease by K/λ Analysis: Correlation With Immunoglobulin Gene DNA Rearrangement

Abstract Previous studies have suggested that analysis of the distribution of surface immunoglobulin light chain isotypes by flow cytometry provides evidence for monoclonality of B cell tumors and may detect populations of circulating tumor cells in patients with lymphoproliferative disease. We have used simultaneous flow cytometry and DNA restriction enzyme analysis on 58 samples of tissue and blood to determine whether lymphocyte populations detected by “kappa/lambda” analysis are indeed monoclonal. In greater than 90% of cases, abnormalities detected by flow cytometry correlated with monoclonal rearrangements of immunoglobulin genes as detected by Southern blot analysis. By analyzing tissue and blood from the same patients, we have also demonstrated that monoclonal circulating cells detected by flow cytometry reflect peripheral circulating tumor cells, since DNA from these cells shows the same immunoglobulin rearrangement as DNA from the original tumors in these patients. Although mixing studies suggested that DNA rearrangement studies were more sensitive than was flow cytometry in detecting minor populations of monoclonal lymphocytes, we found only one case in which this affected the diagnostic accuracy of the kappa/lambda analysis, with one notable exception, that of detection of a monoclonal proliferation of B cells that did not express surface immunoglobulin. The kappa/lambda test thus offers a powerful diagnostic tool in the evaluation of lymphoproliferative disease.

Applications and limitations of peripheral blood lymphocyte immunoglobulin light chain analysis in the evaluation of non-Hodgkin's lymphoma.

Surface immunoglobulin (sIg) light chain analysis of peripheral blood lymphocytes (PBL) from 63 patients with non-Hodgkin's lymphoma (NHL) was performed to determine the ratio of kappa-bearing lymphocytes to lambda-bearing lymphocytes (kappa/lambda ratio). In 43% an abnormal kappa/lambda ratio was detected, implying the presence of a malignant clone in the peripheral blood (clonal excess). In 67% of cases with an abnormal kappa/lambda ratio, the absolute lymphocyte count was within normal limits and 20% did not have morphologic bone marrow involvement. Light chain analysis of bone marrow aspirates was performed in three of four patients with a circulating clone and normal bone marrow morphology. All three patients had abnormal bone marrow aspirate kappa/lambda ratios. The presence of a circulating clone was associated with morphologic bone marrow involvement (P less than 0.05). Nine patients with documented B-cell NHL were followed with repeated examinations. The presence of a circulating clone persisted in two patients refractory to therapy and in two patients otherwise believed to be in remission. Conversion to an abnormal kappa/lambda ratio occurred in two patients coincident with the development of new bone marrow involvement and in a third patient prior to the onset of frank leukemia. In three instances, the PBL light chain analyses remained within normal limits in patients with stable lymphadenopathy. The kappa/lambda ratio returned to normal in one patient responding to treatment with a partial remission, and remained within normal limits despite progression of lymphadenopathy in one patient, and local disease recurrence in another patient. These findings suggest that the detection of a circulating clone by sIg light chain analysis may be useful as a noninvasive approach to identify disseminated disease activity unsuspected on the basis of morphologic evaluations, but may not reflect the presence, progression, or recurrence of localized tissue lesions.

Phenotypic analysis by flow cytometry of surface immunoglobulin light chains and B and T cell antigens in lymph nodes involved with non-Hodgkin's lymphoma

The objective of this study was to demonstrate the diagnostic usefulness of flow cytometric analysis of surface membrane immunoglobulin light chain and monoclonal antibody reactivities in B cell non-Hodgkin's lymphoma. For this purpose, lymph node cell suspensions from 80 patients (20 normal lymph nodes, 11 lymph nodes with benign lymphoid hyperplasia, and 47 lymph nodes with B cell non-Hodgkin's lymphoma) were studied to detect the expression of surface B and T cell differentiation antigens recognized by a panel of monoclonal antibodies (anti-Leu-1, anti-Leu-5, anti-HLA-DR, J-5, anti-BL-1, anti-BL-2, and anti-BL-7). The clonal excess calculation, percent kappa-positive minus percent lambda-positive/percent kappa-positive plus percent lambda-positive cells per discrete level of fluorescence intensity, was used to study the clonality of surface membrane immunoglobulin light chain expression. Among the BL surface antigens, BL-7 proved to be most consistently expressed in B cell non-Hodgkin's lymphoma (79 percent). It was also present in 57 percent of lymph nodes with benign, hyperplasia. No significant relationships were detected between the patterns of reactivity with the anti-BL monoclonal antibodies and histologic subtypes, although the small number of cases tested in each category precludes any definitive conclusions. Immunophenotypic heterogeneity within subgroups was also observed with expression of the other antigens examined. Monoclonal expression of surface membrane immunoglobulin light chain was seen in 43 of 47 (91 percent) of lymph nodes with non-Hodgkin's lymphoma, three of 11 (27 percent) hyperplastic lymph nodes, and one of 22 (4 percent) normal lymph nodes. When the presence of BL-7 and clonal excess was examined as a panel, 83 percent of B cell non-Hodgkin's lymphomas were positively identified, whereas one normal lymph node and no hyperplastic lymph nodes gave positive results. The simultaneous presence of clonal excess and BL-7 can be a useful diagnostic aid in the differentiation of lymphomatous from hyperplastic lymph nodes. Cytofluorimetry provides a rapid, objective, and reproducible technology to confirm the diagnosis of lymph node involvement in B cell non-Hodgkin's lymphoma.

Flow cytometric light chain analysis of peripheral blood lymphocytes in patients with non-Hodgkin's lymphoma.

Peripheral blood lymphocytes from 96 patients with non-Hodgkin's lymphoma were studied, either at primary staging, during treatment or in follow up. The amount of surface immunoglobulin light chain per cell was determined by direct immunofluorescence staining analysed by flow cytometry. Discrepancy between kappa and lambda fluorescence profiles in the sample was considered to indicate the presence of monoclonal cells i.e., circulating lymphoma cells. The results were correlated with routine haematological findings, histopathology of the lymphoma and tumour burden. Using routine haematological methods leukaemic spread was evident in 24% of the patients in our study. Using kappa/lambda distribution analysis evidence of circulating lymphoma cells was found in an additional 27%. As expected, the major diagnostic gain was in the low grade malignant group, where 30% of the patients with normal peripheral blood according to standard procedures showed evidence of circulating lymphoma cells in the kappa/lambda distribution analysis. The corresponding gain in the high grade malignant group was 19%. In patients with active disease but without morphological evidence of leukaemia, 37% showed abnormal kappa/lambda distributions. In patients in complete remission the corresponding figure was 18%. The clinical significance of small numbers of circulating lymphoma cells is not yet understood, but a possible outlook is to use kappa/lambda distribution analysis to increase staging precision and in the early detection of relapse.

The cellular content of non Hodgkin lymphomas: a comprehensive analysis using monoclonal antibodies and other surface marker techniques.

Five samples of tonsil, 10 reactive lymph nodes and 65 consecutive cases of non-Hodgkin lymphoma (NHL) were evaluated in suspension phenotyping with the monoclonal antibodies alpha Leu-I, alpha Leu-2a, alpha Leu-3a, OKT1, OKT3, OKT4, OKT6, OKT8, W6/32, 26/114, DA-2, 2DI, J5, AN51 and OKT9 together with conventional surface marking by rosetting (E, Fc gamma, Fc mu, C3b, C3d) and staining for surface and cytoplasmic immunoglobulin (SIg, CyIg) heavy and light chain classes. The results confirm the reproductability and specificity of staining with monoclonal antibodies against T cells and T cells subsets. Evidence is presented for reactivity of alpha Leu-I antibody with SIg positive and Ia positive cells in some lymphomas (centroblastic centrocytic, lymphocytic and immunoblastic), and 2 cases showed evidence of marking with OKT3 on SIg positive cells in T cell predominant immunoblastic lymphoma. Lymphoblastic lymphomas of T cell type expressed the marker OKT6. On the basis of these results criteria for the diagnosis of T cell lymphoma are suggested. The monoclonal antibody J5, reactive with C-ALL antigen, showed variable positivity, occasionally strong in B cells in cases of centroblastic and centrocytic follicular lymphoma. Proportions of cells staining with the monoclonal antibody OKT9 showed a correlation between levels of cellular expression of transferrin (trf) receptor and the histological grade of malignancy, OKT9+ cells being elevated in high grade lymphomas, and in some cases of transforming lymphoma of low grade histological class. These results are discussed and indicate the advantage of employing a wide range of defined monoclonal reagents in the phenotypic evaluation of NHL.

The immunologic characterization of 40 extranodal lymphoid infiltrates: usefulness in distinguishing between benign pseudolymphoma and malignant lymphoma.

In the studies described here, 40 extranodal lymphoid tumors obtained from 38 patients were evaluated by cell-marker analysis and the results correlated with the light microscopic features. These infiltrates were investigated for the present composition of cells expressing Ia antigens, surface immunoglobulin (SIg), including kappa and lambda light chains, sheep erythrocyte (E) rosette formation, and acid a-naphthyl acetate esterase (ANAE) activity. Fifteen biopsy specimens consisted of variable proportions of benign T and polyclonal B cells; these 15 lesions had the histopathologic features of benign pseudolymphomas. The remaining biopsy specimens consisted almost entirely of B cells bearing monoclonal SIg (18 cases) or a great preponderance of T cells (five cases) or non-B, non-T (null) cells (two cases); these 25 lesions were classified histopathologically as malignant lymphomas. Thus, the extranodal lymphoid infiltrates were divisible, according to their cell-marker characteristics, into two categories: lesions that are immunologically polyclonal and lesions that are immunologically monoclonal B-cell proliferations or consist of a great preponderance of T or null cells. In each case, polyclonality correlated with benign cytomorphologic features and monoclonality correlated with malignant histopathology. Cell-marker analysis appears to represent an important adjunct to light microscopy in distinguishing histologically problematic benign pseudolymphomas from malignant lymphomas that arise in the extranodal tissues. Cell marker analysis will undoubtedly provide insights into the histogenesis, natural history, and biologic behavior of the extranodal lymphoid neoplasms not attainable using light microscopy alone.

Colony-forming assay for circulating chronic lymphocytic leukemia cells

In these studies, we report adaptation of a colony-forming assay to chronic lymphocytic leukemia (CLL) peripheral blood cells. T-lymphocyte-depleted CLL peripheral blood cells were cultured with irradiated, normal T cells and media conditioned by normal, mitogen-stimulated T cells in methylcellulose. Colonies containing small and transformed lymphocytes appeared after 5–7 days incubation. The plating efficiency of CLL colonies was 0.15 ± 0.08% ( x ± S.D. ), similar to that of other colony-forming systems. The majority of CLL colony-forming cells were in S phase (50 ± 4%, x ± S.E. ) as determined by thymidine suicide and the fraction of colony-forming cells in S phase was inversely related to the WBC. Cells harvested from CLL colonies lacked surface markers for T lymphocytes and stained positively for monoclonal surface and/or cytoplasmic immunoglobulin light chains. A 1-h incubation was used to study the in vitro response of CLL colony-forming cells to adriamycin and melphalan. Preliminary studies suggest differences in patterns of in vitro sensitivity to melphalan between patients previously treated with alkylating agents and those who had not received treatment. This system can be used to study regulation of CLL cell proliferation, and may have utility in predicting response to chemotherapeutic agents.

Surface immunoglobulins on thymus and thymus-derived lymphoid cells.

Lymphoid cells from thymus, thoracic duct lymph (TDL), and thoracic duct lymph in irradiated animals reconstituted with allogeneic thymus cells (TTDL) were labeled with radioiodinated anti-immunoglobulins using radioautographic techniques. Thymus and TTDL were labeled (14.4 and 37.0%, respectively) with anti-light chain protein after prolonged exposures (30-60 days). No labeling was observed on thymus and TTDL with anti-polyheavy chain globulin. In contrast 18.5-19.0% of TDL labeled on short exposure (6 days) with anti-polyheavy chain and anti-light chain materials. It is proposed that the difference between the labeling observed on short exposures versus long exposures can be related to the difference in surface density of immunoglobulins between nonthymus-derived (B) and thymus-derived (T) cells. The distribution of labeled cells in the thymus was preferentially among the larger cells (greater than 10 micro diameter). The TTDL population was mostly composed of a larger, blast-like population and the distribution of label was independent of size. As the thymus and TTDL preparations contain almost exclusively T cells, this represents a direct demonstration of surface immunoglobulin light chains on T lymphoid cells.

Surface immunoglobulin light chain determinants on normal and pha-stimulated human blood lymphocytes studied by immunofluorescence and electronmicroscopy

Lymphocytes from human peripheral blood were cultured in vitro. The distribution of antigenic determinants with specificity of immunoglobulin light or heavy chains on normal and phytohaemagglutinin (PHA) stimulated cells was examined by membrane immunofluorescence and immunoelectron microscopy. It appeared that a relatively large population of both normal and stimulated lymphocytes carried light chain determinants on their surface. Usually considerably more lymhocytes reacted for ϰ than for λ determinants. In contrast no or only few cells reacted with reagents specific for heavy chain (γ or μ) determinants. At the ultrastructural level, the reactivity to antilight chain antibodies in unstimulated cultures was seen to be confined to discontinuous areas of varying size on the surface of cells with the typical morphology of small to medium sized lymphocytes. In cultures treated with PHA, lymphocytes undergoing blastoid transformation often showed larger patches of antilight chain reactivity than unstimulated lymphocytes. The uropods of stimulated lymphocytes often showed a strong reactivity with antilight chain antibodies. No reactions were obtained with antibodies to heavy chains. In contrast, normal as well as PHA-stimulated lymphocytes stained for human species antigens by means of heterologous antisera from rabbits, exhibited an even distribution of the ferritin label over large areas of their surface.