Expression of the Leu-8 antigen by B-cell lymphomas

The Leu-8 antigen is found on the surface of many hematologic cells, including many T- and B-lymphocytes. With the use of a frozen-section immunoperoxidase technic, 152 B-cell non-Hodgkin's lymphomas were examined for Leu-8 expression. Of these lymphomas, 53% expressed Leu-8. Subclassification of the lymphomas with the use of the International Working Formulation showed that most small lymphocytic, intermediate lymphocytic, and diffuse large cell lymphomas and about half of diffuse small cleaved, diffuse mixed, and follicular lymphomas expressed Leu-8. In contrast, all 17 cases of small noncleaved cell (Burkitt's) lymphoma and 9 of 10 cases of multiple myeloma/plasmacytoma were Leu-8 negative. These results indicate that Leu-8 is expressed on a wide variety of B-cell lymphomas and that differences in Leu-8 expression may be useful in the diagnostic separation of small lymphocytic lymphoma with plasmacytoid features from multiple myeloma/plasmacytoma, and diffuse large cell lymphoma from Burkitt's lymphoma.     

Expression of Leu-8 surface antigen in B-cell lymphomas. Correlation with other B-cell markers

Using in situ immunohistological analysis, expression of Leu-8 and its correlation with other B-cell markers were investigated in 21 selected lymphomas of different categories, each one expressing its own typical immunophenotype. These categories included eight follicular centroblastic/centrocytic (CB/CC) lymphomas, eight intermediately differentiated lymphocytic lymphomas (ILL)/mantle zone lymphomas (MZL), and five lymphocytic lymphomas (LL) associated with chronic lymphocytic leukaemia (CLL). Four reactive lymph nodes and three tonsils were also studied using double immunolabelling procedures. Cell suspensions were also performed in three CB/CC and four ILL/MZL cases. Leu-8 was consistently expressed in ILL/MZL and LL but it was absent in most (7/8) CB/CC lymphomas. In reactive tissues, the Leu-8-positive B cells were strictly confined to the mantle zones. A close association emerged between Leu-1 (CD5) and Leu-8, both being present in ILL/MZL and LL but absent in CB/CC. A consistent lack of association was found between Leu-8 or CD5 antigens and common acute lymphoblastic leukaemia antigen (CD10) and BA-2 (CD9) antigen, whereas Leu-8 and CD5 were strictly associated with surface IgD. Reactivity with Leu-8 provides a means of distinguishing between CB/CC and ILL/MZL. Furthermore, shared immunoreactivity for Leu-8 in ILL/MZL and LL may represent a potential clue to the still uncertain cellular derivation of LL/B-CLL.     

Small B-cell lymphoid neoplasms with coexisting T-cell lymphomas.

The simultaneous occurrence of a small B-cell lymphocytic neoplasm and a T-cell lymphoma in the same lymph node biopsy specimen is documented in two patients. The biopsy from the first patient, who had a 5-year history of chronic lymphocytic leukemia, showed evidence of a small B-cell lymphocytic neoplasm coexisting with a large-cell lymphoma of T-cell phenotype. The lymph node biopsy specimen from the second patient showed features of small lymphocytic lymphoma of B-cell phenotype, coexisting with a small-cell pleomorphic lymphoma of T-cell phenotype. The lymph node specimens from both patients met strict criteria for composite lymphomas. The clinical and morphologic findings in the first patient are those of "Richter's transformation" of chronic lymphocytic leukemia. The lymph node biopsy specimens from these two patients demonstrate that small B-cell lymphocytic neoplasms may coexist with T-cell lymphomas.     

DRC antigen expression in B-cell lymphomas

DRC (dendritic reticulum cell) antigen expression was studied in 38 cases of B-cell lymphomas including follicular lymphoma. The results of this study showed DRC-1 to be expressed in 1/3 of small lymphocytic; 3/3 of mantle zone lymphoma (MZL); 10/10 of follicular, small cleaved; 6/7 of follicular, mixed; 1/2 of follicular, large cell lymphomas. However, DRC-1 was not expressed in any of diffuse, small cleaved (0/6) and diffuse, large cell (0/6). Although S-100 protein was positive in the majority of these DRC-1-positive cases on the paraffin embedded specimens, positive nodules were less intense and smaller in number compared with those of DRC-1 on frozen tissue specimens. These results suggest that in case of small lymphocytic lymphoma found to be positive for DRC-1, the networks of the DRCs are expressed in the pseudofollicular proliferation centers. This study also suggests that the networks of the DRCs newly appear accompanying the neoplastic growth rather than originating from residual germinal centers, and that neoplastic small cleaved cells play a major role in inducing the DRCs in the positive cases of follicular lymphoma, MZL, and small lymphocytic lymphoma.     

Expression of Tac antigen by non-Hodgkin's lymphomas

Anti-Tac is a monoclonal antibody that appears to recognize the interleukin-2 receptor. With the use of a frozen-section immunoperoxidase technic, a large series of non-Hodgkin's lymphomas were investigated for the presence of Tac-antigen on neoplastic cells. Approximately one-fourth of cases expressed the Tac antigen, including 27% of B-lineage lymphomas, 6% of the T-lineage lymphomas, and three of four cases of Ki-1-expressing lymphoma. The B-lineage lymphomas with the highest incidence of Tac antigen expression were the large cell lymphomas, both diffuse and follicular, where about one-half of cases expressed the Tac antigen. All major categories of lymphoma expressed Tac except plasma-cytoma/myeloma, small noncleaved cell (Burkitt's and non-Burkitt's), and lymphoblastic malignancies.     

Low-grade lymphomas. Expression of developmentally regulated B-cell antigens

A series of low-grade B-cell lymphomas was analyzed for a battery of immunologic determinants by flow cytometry and immunohistochemistry. Histologically distinctive subclasses of these lymphomas, well-differentiated lymphocytic (WDL), intermediately differentiated lymphocytic (IDL), and follicular center cell (FCC) lymphoma, were found to be readily distinguishable by their expression of immunologic determinants that are known to be developmentally regulated in normal B cells. Although all cases expressed monoclonal surface immunoglobulin (sIg), HLA-DR, and the surface membrane proteins recognized by antibodies B1 (p32) and BA1, staining with other monoclonal antibodies revealed unique immunologic phenotypes for each subclass: WDL p65 (Leu 1)+, p24 (BA2)-; IDL p65+, p24+; FCC p65-, p24-. Additionally, the fluorescence intensities (number of determinants per cell) obtained for sIg, BA-1, and B1, but not HLA-DR, were significantly different among the three lymphoma subclasses. The relative fluorescence intensities of each of these three markers followed the same pattern: FCC greater than IDL greater than WDL. Taken together, these distinguishing features suggest that low-grade B-cell lymphomas represent arrested, and possibly sequential, stages of B-cell differentiation.     

Immunoregulatory Leu-7+ and T8+ lymphocytes in B-cell follicular lymphomas

Lymphocyte subpopulations were profiled in lymph nodes and tonsils showing follicular hyperplasia and in follicular lymphomas with monoclonal antibodies on frozen tissue sections. Immunoregulatory lymphocyte subsets identified with T8 and Leu-7 monoclonal antibodies were quantified within the follicular centers (FC) of the nonneoplastic tissue and neoplastic follicles of the lymphomas with an optical grid defining a unit surface area (USA) of 0.04 mm2. T8+ cells were essentially confined to the interfollicular areas, with a few cells occupying the FC of the nonneoplastic specimens (mean, two and five cells/USA for tonsils and benign lymph nodes, respectively). Although lymphomas exhibited a similar pattern of distribution of T8+ cells, 17 T8+ cells/USA were observed in the follicular small cleaved cell (FSCL) group and eight T8+ cells/USA within the follicular mixed small cleaved and large cell (FML) group. Leu-7+ cells were almost entirely confined to the FC of the nonneoplastic tissues and increased (mean, 17 and 19 cells/USA for tonsils and benign lymph nodes, respectively) compared with the T8+ population. Variable distributions of Leu-7+ cells were found in the FSCL group, with a mean of 16 cells/USA. Very few Leu-7+ cells were present in the FML group. Natural killer cells and/or cytotoxic/suppressor T lymphocytes may play an immunoregulatory role in modulating the growth of follicular lymphomas.     

Expression of T-cell receptor (TCR)alpha chain on normal human tonsils and T-cell lymphomas

We analyzed immunohistologically the expression of T-cell receptor (TCR)alpha chain on human tonsils and on T-cell lymphoma (T-ML) tissues using the avidin-biotin-peroxidase method. A murine monoclonal antibody alpha F1, specific for the constant region of the TCR alpha chain, was employed. On normal tonsil, alpha F1-positive cells were observed mainly in T-zones and germinal centers. In T-zones, the staining intensities varied markedly, with heavy staining evident in less than one fourth. In germinal centers, a proportion of stained cells showed a histiocytic pattern with small cytoplasmic projections. All the T-ML tissues expressed TCR alpha, whereas the staining intensities varied among cases and among lymphoma cells. No correlations were observed in the expressions of TCR alpha chain and other T-cell markers including CD3, CD4 and CD8.     

Studies on B-cell memory. II. T-cell independent antigen can induce B-cell memory.

Both athymic nude mice and normal mice primed with a T-cell independent antigen, i.e. dinitrophenylated dextran (DNP-DE), at a sub-immunogenic dose, produced very poor anti-DNP responses to a later challenge with the same antigen. B-cell memory was expressed, however, as an enhanced IgM response after the challenge of the DNP-DE-primed mice with the T-cell dependent antigen (dinitrophenylated haemocyanin, DNP-KLH) in the presence of functional T-cells. Moreover, DNP-DE-primed spleen cells also revealed an enhanced IgM response after adoptive transfer into irradiated recipients and challenge with DNP-DE. The injections of DNP-DE-primed nude mouse serum into unprimed mice resulted in the reduction of anti-DNP response to the immunization with DNP-DE. These results indicate that (a) T-cell independent DNP-DE causes the differentiation of B cells not only into antibody-forming cells but also into memory cells, (b) these memory cells can be triggered in situ by the T-cell dependent DNP-KLH in the presence of helper T cells but not by T-cell independent antigen, and (c) some humoral factor(s) induced by DNP-DE-priming seems to interfere with the expression of B-cell memory only when challenged with T-cell independent DNP-DE.     

Leu-22 (L60). A more sensitive marker than UCHL1 for peripheral T-cell lymphomas, particularly large-cell types

Paraffin-embedded sections of 77 peripheral T-cell lymphomas (PTCLs) were stained with several monoclonal antibodies, including the preferential T-cell markers Leu-22 (L60[CD43]) and UCHL1 (CD45RO). The staining characteristics of L60 and UCHL1 were compared to determine the value of each in the immunophenotypic analysis of PTCLs. Lineage specificity was evaluated among 39 B-cell lymphomas and 33 cases of Hodgkin's disease (HD). L60 and/or UCHL1 stained 95% of PTCLs, whereas L60 and UCHL1 alone stained 90% and 69% of cases, respectively. L60 demonstrated significantly greater numbers of immunopositive tumor cells than UCHL1 in 37% of the PTCL cases, principally because of enhanced marking of large, neoplastic cells. UCHL1 was a better marker in only 10% of the PTCL cases. L60 stained 33% of B-cell lymphomas, usually small lymphocytic or lymphoplasmacytic types. UCHL1 stained only 8% of B-cell lymphomas, all large-cell types. L60 and UCHL1 stained Reed-Sternberg cells and variants in three cases of nodular sclerosing HD. These results suggest that both L60 and UCHL1 are useful markers of PTCLs in routinely processed tissue. L60 is a more sensitive marker of large neoplastic T-cells than UCHL1 but is less lineage-specific. These antibodies are most effective when used as part of a panel of monoclonal antibodies.     

Activated T-cell subsets in benign lymphoid hyperplasias and B-cell non-Hodgkin''s lymphomas.

Activated tumor-infiltrating T lymphocytes (TIL-T) were quantitated prospectively in excisional biopsy specimens of 49 B-cell non-Hodgkin's lymphomas (NHL) of various grades and compared with eight benign lymphoid hyperplasias (BLH) to identify any potential difference in host T-cell response. Immunotyping of tissue-cell suspensions was done by three-color flow cytometry, which was complemented by immunocytology by using cytocentrifuged preparations. Two activated T-cell subsets were studied: acutely activated TIL-T (CD3+ CD25+ HLADr-) and chronically activated TIL-T (CD3+ CD25- HLADr+). Results showed an association of the more aggressive intermediate/high-grade B-cell NHL with a higher percentage of late-phase activated TIL-T and a progressive increase with the grade of malignancy: 10.29%, 23.25%, 33.87%, and 47.78% (means) for BLH and for low-, intermediate- and high-grade B-cell NHL, respectively. Differences for this subset were significant (P less than 0.050) for the following comparisons: hyperplasia versus intermediate-grade NHL (P less than 0.0012), hyperplasia versus high-grade NHL (P less than 0.0002), and low versus high-grade NHL (P less than 0.0080). The percentage of acutely activated TIL-T cells did not show a statistically significant difference between the groups. The results suggest a host T-cell response to proliferating neoplastic cells in B-cell NHL. Paradoxically, the response does not appear to be protective for the host since the intensity is directly proportional to the grade of malignancy. However, the recognition of this response may have clinical applications since its amplification with biological response modifiers may result in effective adoptive immunotherapy of B-cell NHL. Further clarification of the specificity and biologic significance of host T-cell activation in B-cell NHL will require functional studies of isolated lymphocytic subpopulations from neoplastic tissue.     

Distribution of T-cell subsets in follicular and diffuse lymphomas of B-cell type.

The authors examined the number and distribution of cells reacting with monoclonal antibodies to T-cell subsets in frozen tissue sections of B-cell lymphomas (30 follicular and 17 diffuse lymphomas). In five diffuse lymphomas (two lymphocytic, three small cleaved cell) the neoplastic B-lymphocytes reacted with the monoclonal antibody anti-T1. In all other cases, the monoclonal antibodies to T-cell subsets reacted only with small lymphocytes concentrated between the follicles of follicular lymphomas and distributed randomly in diffuse lymphomas. The distribution of T cells and the T4+/T8+ ratio in follicular small cleaved and mixed lymphomas was similar, although not identical, to that seen in hyperplastic lymphoid follicles. Fewer T cells and a decrease in the T4+/T8+ ratio were seen in follicular large cell lymphoma and in diffuse large cell lymphomas. The number and distribution of T cells in follicular lymphomas is consistent with the hypothesis that there is a functional interaction between neoplastic B cells and benign T cells. No tumors were found in which the neoplastic B cells reacted with anti-T3, anti-T4, or anti-T8.     

Expression of Tac antigen in B cell lymphomas.

In a series of 55 cases of B cell derived non-Hodgkin's lymphoma the reactivity of two distinct anti-Tac monoclonal antibodies was examined using a sensitive immunoperoxidase technique on cryostat sections. Eighteen out of the thirty-five cases of B cell lymphomas of low or intermediate grade of malignancy were found to be reactive while six out of 20 cases of high-grade malignancy lymphomas showed a positive immunostaining. No correlation was found between anti-Tac reactivity and surface immunoglobulin phenotype, T65 antigen, or calla expression. These findings showed that IL2 receptor expression is not restricted to activated T cells, and raise the question of the possible role of IL2 in the regulation of malignant B cell clone expansion.     

Expression of Grb2 distinguishes classical Hodgkin lymphomas from primary mediastinal B-cell lymphomas and other diffuse large B-cell lymphomas

Growth factor receptor-bound protein 2 is an adaptor molecule that mediates B-cell receptor (BCR) signaling pathways, but the expression of growth factor receptor-bound protein 2 in lymphoma tissues has not been reported. We sought to characterize growth factor receptor-bound protein 2 protein expression in reactive tonsillar tissues and lymphoma tissues obtained from diagnostic biopsies of classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and 20 low-grade B-cell lymphomas. Growth factor receptor-bound protein 2 expression was assessed in tissues by immunohistochemistry and in lymphoma cell lines by immunoblotting. In reactive lymphoid tissues, growth factor receptor-bound protein 2 was expressed in the cytoplasm of B-cells and histiocytes but not T-cells. Strong, cytoplasmic growth factor receptor-bound protein 2 expression was seen in the neoplastic cells of follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma, splenic marginal zone lymphoma, primary mediastinal large B-cell lymphoma, diffuse large B-cell lymphoma, and nodular lymphocyte predominant Hodgkin lymphoma. In contrast, only 10% of the classical Hodgkin lymphomas showed growth factor receptor-bound protein 2 expression in the neoplastic cells. Growth factor receptor-bound protein 2 protein expression was detected by Western blotting in all lymphoma cell lines tested with higher levels in primary mediastinal large B-cell lymphoma compared with classical Hodgkin lymphoma cell lines. These findings support a role for growth factor receptor-bound protein 2 in the diagnostically challenging workup of classical Hodgkin lymphoma versus primary mediastinal large B-cell lymphoma and warrant further studies to evaluate the biologic significance of growth factor receptor-bound protein 2 in the pathogenesis of classical Hodgkin lymphoma.     

Expression of TCL1 and CD27 in primary cutaneous B-cell lymphomas

AIMS: To investigate by immunohistochemical analysis the expression of the TCL1 oncogene product and of CD27 in 25 cases of primary cutaneous B-cell lymphomas (PCBCL) classified according to the World Health Organization-European Organization for Research and Treatment of Cancer classification of cutaneous lymphomas. In B-cell ontogenesis TCL1 is mainly expressed by 'naive' B lymphocytes and by a subset of germinal centre B cells, whereas CD27 is expressed by a subset of germinal centre B cells, 'memory' B lymphocytes and plasma cells, suggesting that their expression in physiological conditions is mutually exclusive. METHODS AND RESULTS: Overall, TCL1 was expressed in 5/25 cases (20%) and CD27 in 15/25 cases (60%). Furthermore, 7/25 cases (28%) were TCL1- and CD27- and 2/25 cases (8%) were TCL1+ and CD27+. In particular, primary cutaneous follicle-centre lymphomas (10 cases) showed a variable expression of both TCL1 and CD27, whereas primary cutaneous marginal-zone B-cell lymphomas (eight cases) showed, with the exception of a single case, a definite CD27+/TCL1- profile. CONCLUSIONS: These findings indicate: (i) the TCL1 oncogene product is uncommonly expressed in PCBCL (20% of cases, mainly of the follicle-centre subtype); (ii) in contrast, CD27 is often expressed in PCBCL (60% of cases), mainly of the marginal-zone subtype; (iii) the coexpression of TCL1 and CD27 may be seldom observed in PCBCL (8% of cases); (iv) PCBCL does not seem to show, in terms of either TCL1 or CD27 expression, significant differences compared with its systemic counterparts.     

Studies on B-cell memory. I. Generation and exhaustion of B-cell memory by thymus-dependent antigen in T-cell depleted mice.

Mice depleted of T cells by adult thymectomy, X-irradiation and reconstitution with syngeneic bone marrow cells, either untreated or treated with anti-Thy-1 serum and complement, were immunized intensively with alum-precipitated bovine serum albumin (AP-BSA) along with or without bacterial lipopolysaccharide (LPS), but no significant anti-BSA antibody response was detected. Priming of the T-cell depleted mice, however, either by a single injection of AP-BSA plus LPS or by multiple injections of AP-BSA without LPS, resulted in the generation of immunological memory. A single injection of AP-BSA without LPS was ineffective. The memory required the aid of syngeneic T cells to be recalled by the challenge with AP-BSA plus LPS. On the other hand, multiple injections of AP-BSA plus LPS did not cause the generation of memory and the response of these mice to the challenge was lower than that of unprimed control mice. These results suggest that (1) the anti-BSA response is highly dependent on the helper function of T cells, (2) the degree of T-cell requirement for the memory generation is very low, and (3) priming with too much strong stimulation in the absence of functional T cells leads to the suppression or abortion of previously generated immunological memory.     

AIDS and non-AIDS diffuse large B-cell lymphomas express different antigen profiles.

Based on gene expression profiling, diffuse large B-cell lymphomas arising in immunocompetent patients can be divided into germinal center and activated B-cell types. Since little is known about acquired immunodeficiency syndrome associated diffuse large B-cell lymphomas, we tested whether the protein expression of germinal center and activated B-cell markers differed between acquired immunodeficiency syndrome (AIDS) vs non-AIDS diffuse large B-cell lymphomas. We immunohistochemically stained tissue microarrays of 39 de novo diffuse large B-cell lymphomas: 12 AIDS associated and 27 non-AIDS, with germinal center (BCL6, CD10, CyclinH) and activated B-cell markers (MUM1, CD138, PAK1, CD44, BCL2). We scored each case for percent positive cells (0-19%=0; 20-49%=1; 50-100%=2). The activated B-cell and germinal center summation scores of each case were used as (x, y) coordinate data points to construct two-dimensional contour-frequency plots. The contour plot of non-AIDS diffuse large B-cell lymphomas showed two distinct clusters: a cluster with a high germinal center phenotype (cluster 1) and a cluster with a high activated B-cell phenotype (cluster 3). In contrast, the AIDS-related diffuse large B-cell lymphomas formed a single aggregate (cluster 2) (P=0.02, Fisher exact test). When the contour plots of the AIDS-related and the non-AIDS cases were superimposed, cluster 2 of the AIDS cases expressed an intermediate germinal center/activated B-cell phenotype compared to clusters 1 and 3 of the non-AIDS diffuse large B-cell lymphomas. Our results confirm that non-AIDS diffuse large B-cell lymphomas segregate into two groups with either germinal center or activated B-cell phenotype. We report the new finding that the AIDS status of the patient predicts the immunophenotype of the diffuse large B-cell lymphomas.     

The Leu-1 B-cell subpopulation in patients with rheumatoid arthritis

Ly-1- B cells have been shown to be elevated in autoimmune mice, especially NZB, and to secrete IgM autoantibody, suggesting that Ly-1 B cells may participate in autoimmune diseases. In this study, B-cell populations carrying Leu-1, a human T-cell surface molecule homologous to mouse Ly-1 (Leu-1 B), were examined by dual fluorescence flow cytometry, in peripheral blood lymphocytes taken from patients suffering from various autoimmune disease. These B cells were shown to be present at a significantly high percentage in rheumatoid arthritis (RA) patients. There was no significant correlation among the incidence of Leu-1 B cells, rheumatoid factor (RF) titers, and the amount of IgM productionin vitro. There was, however, a significantly high incidence of Leu-1 B cells in RA patients with a high RF titer (greater than 5 × 2¹³), suggesting that these B cells in RA may play an important role in IgM RF productionin vivo.     

Expression of the low Mr isoform of CD45 (CD45RO) in B-cell non-Hodgkin's lymphomas.

The CD45 antigen family consists of multiple molecular isoforms ranging from 180 to 220 relative molecular mass (Mr). The highest Mr isoforms are recognized by monoclonal antibodies (MoAbs) designated CD45RA, while those recognizing the low Mr isoform are designated CD45RO. About half of the T-cells in peripheral blood express CD45RA while the remainder express CD45RO. A switch from the high to the low Mr isoform of CD45 has been found in association with the process of T-cell stimulation and acquisition of "memory." B-cells normally express CD45RA, but not CD45RO. However, under stimulatory conditions, B-cells may be capable of undergoing an isoform switch and expressing CD45RO. The expression of this low Mr isoform of CD45 was investigated in lymphomas composed of monoclonal B-cells to determine if such a switch occurs in malignant B-cell populations. The vast majority (110/117 cases) of B-cell lymphomas expressed only CD45RA, while a very small number (7/117 cases) expressed CD45RO, but not CD45RA. There was no relationship between the CD45RO expression and the histologic subtype. The physiological significance of this unusual expression of CD45RO in a subpopulation of B-cell lymphomas is not clear. In that CD45RO, as defined by the MoAb UCHL 1, is typically used as a marker of T-cells in tissue sections, caution must be exercised in interpretation, since not all T-cells are reactive and some B-cell lymphomas are reactive.