Elevated serum levels of soluble CD23 (sCD23) precede the appearance ofacquired immunodeficiency syndrome--associated non-Hodgkin's lymphoma

Human immunodeficiency virus (HIV) infection-associated B-cell hyperstimulation, in particular, the chronic stimulation of B cells to undergo isotype switching, may play an important role in the pathogenesis of acquired immunodeficiency syndrome-associated lymphoma (AIDS lymphoma). Isotype switching can be induced by various immune system factors, including cytokines, cell-surface stimulatory molecule interactions, and CD23. CD23 is a B-cell differentiation and activation marker expressed on mature B cells that is lost after isotype switching; soluble CD23 (sCD23) also is a B-cell-stimulatory factor. Because sCD23 is associated with Ig isotype switching, and because an enhancement of isotype switching may contribute to the genesis of AIDS lymphoma, we examined serum sCD23 levels in a retrospective study of HIV-seropositive subjects who had gone on to develop lymphomas. Subjects were participants in the Multicenter AIDS Cohort Study at UCLA, a study of the natural history of AIDS. Greatly elevated sCD23 serum levels were seen in subjects who developed AIDS lymphoma, when compared with others with AIDS (without lymphoma), or to HIV- seronegative or HIV-seropositive subjects who did not have AIDS. Because the induction of IgE has been tied to the activity of CD23, serum IgE levels were also examined in this study, and found to be significantly elevated in those who developed AIDS lymphoma. These findings suggests that serum sCD23 levels potentially may serve as a clinical tool for early detection of lymphomas in people who have HIV infection. Also, these observations provide clues on possible pathogenetic mechanisms that result in lymphomagenesis in the context of HIV infection and AIDS.     

Responsiveness of chronic lymphocytic leukemia B cells activated via surface Igs or CD40 to B-cell tropic factors.

Recent studies performed in the laboratory have established that interleukin-4 (IL-4) used in combination with anti-CD40 monoclonal antibody (MoAb) 89 presented on Ltk- mouse fibroblasts stably expressing human Fc gamma RII/CDw32 (referred to as the CD40 system) sustains long-term proliferation of normal human B cells. In the present study, B-cell chronic lymphocytic leukemias (B-CLLs) activated through slgs or CD40 were examined for their capacity to proliferate and differentiate in response to various cytokines. Our results indicate that the outcome of IL-4 stimulation on the in vitro growth of B-CLL depends on the signalling pathway used for their activation. Whereas IL-4 did not display any growth-stimulatory effect on B-CLL activated by Ig cross-linking agents, it could stimulate DNA synthesis and enhance the viable cell recovery when leukemic B cells were cultured in the CD40 system. Most B-CLL samples were induced for IgM synthesis upon Staphylococcus aureus strain Cowan I stimulation. This Ig response was potentiated by IL-2 and antagonized by IL-4. Anti-CD40 MoAb used alone or in combination with cytokines (IL-1 alpha to IL-6, interferon gamma, tumor necrosis factor gamma, and transforming growth factor beta) failed to induce Ig secretion from B-CLL cells. No evidence for Ig isotype switching was obtained with the cytokines listed above, regardless of the mode of activation. Taken together, our results suggest that B-CLL cells can be partially released from their apparent maturation block by IL-2 and Ig cross-linking agents. In contrast, combinations of IL-4 and cross-linked anti-CD40 antibodies induced entry of B-CLL cell into cycle, but poorly stimulated their differentiation into Ig secreting cells.     

Induction of IgA1 and IgA2 production in immature human fetal B cells and pre-B cells by vasoactive intestinal peptide

We studied the effects of vasoactive intestinal peptide (VIP) on IgA1 and IgA2 production in human fetal B cells and pre-B cells derived from bone marrow. VIP induced IgA1, IgA2, and IgM production in sIgM+, CD19+ fetal B cells stimulated with anti-CD40 monoclonal antibody (MoAb) without inducing the production of IgG1, IgG2, IgG3, IgG4, or IgE. The anti-CD40 MoAb plus VIP also induced IgA1, IgA2, and IgM production in sIgM-, CD19+ pre-B cells, which was enhanced by the addition of interleukin-7 (IL-7). This induction by VIP was specific, as the anti- CD40 MoAb plus other neuropeptides [ie, somatostatin (SOM) or substance P (SP)] had no effect, and moreover, the induction was specifically blocked by a VIP antagonist. Furthermore, the anti-CD40 MoAb plus various cytokines, including IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, transforming growth factor beta (TGF-beta), low-molecular-weight B-cell growth factor (BCGF), and interferon-gamma (IFN-gamma), did not induce IgA1 and IgA2 production in fetal B cells or pre-B cells. These findings indicate that, in the presence of costimulators, VIP may induce IgA1 and IgA2 production by isotype switching.     

Temporal association of CD40 antigen expression with discrete stages of human B-cell ontogeny and the efficacy of anti-CD40 immunotoxins against clonogenic B-lineage acute lymphoblastic leukemia as well as B-lineage non-Hodgkin's lymphoma cells.

Detailed immunophenotypic analyses of immunologically classified leukemias and lymphomas showed that CD40 displays an exquisite B-lineage specificity within the human lymphopoietic system. Notably, 82% of B-lineage chronic lymphocytic leukemias (CLLs), 82% of B-lineage hairy cell leukemias (HCLs), 86% of B-lineage non-Hodgkin's lymphomas (NHLs), and 29% of B-lineage acute lymphoblastic leukemias (ALLs) were CD40+. Quantitative analyses of the correlated expression of CD40 and other B-lineage differentiation antigens on fetal lymphoid precursor cells by multiparameter two-color/three-color flow cytometry, combined with analyses of sequential antigen expression on fluorescence-activated cell fluorescence activated cell sorter (FACS) isolated immunologically distinct fetal B-cell precursor subpopulations during in vitro proliferation and differentiation, provided evidence that the acquisition of CD40 antigen in human B-cell ontogeny occurs subsequent to the expression of CD10 and CD19 antigens but before the surface expression of CD20, CD21, CD22, CD24, and surface immunoglobulin M (sIgM). Some leukemic pro-B cells from ALL patients as well as normal pro-B cell clones from fetal livers displaying germline Ig heavy chain genes were CD40+, indicating that the acquisition of CD40 antigen likely precedes the rearrangement of Ig heavy chain genes. CD40+ FACS-sorted malignant cells from B-lineage ALL as well as B-lineage NHL patients were capable of in vitro clonogenic growth, indicating the CD40 antigen is expressed on clonogenic leukemia and lymphoma cells. This hypothesis was confirmed by the ability of an anti-CD40 immunotoxin that we used as an antigen-specific cytotoxic probe to effectively kill clonogenic B-lineage ALL and NHL cells.     

Human Lyb-2 homolog CD72 is a marker for progenitor B-cell leukemias.

S-HCL 2 is the prototype antibody of the recently defined CD72 cluster (human Lyb-2). Under nonreducing conditions, S-HCL 2 monoclonal antibody (mAb) precipitates a glycoprotein of 80-86 kDa. Under reducing conditions, a dimer of 43 and 39 kDa, with core proteins of 40 and 36 kDa, is precipitated. CD72 expression in normal and malignant tissues is different from expression of all other previously described human B-cell antigens. In peripheral blood and bone marrow, the antigen appears to be present on all B lymphocytes, with the exception of plasma cells. In tissue, immunohistochemical staining revealed positivity for all known B-cell compartments; however, pulpa macrophages of the spleen and von Kupffer cells exhibited distinct positivity for CD72 also. Among 83 malignant non-Hodgkin's lymphomas examined by immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase technique), all 54 B-cell lymphomas, including precursor B-cell lymphomas, Burkitt's lymphomas, germinal center lymphomas, chronic lymphocytic leukemias, and hairy cell leukemias, were CD72 positive, but no T-cell lymphomas were. Flow cytometry study of more than 80 mainly acute leukemias (52 B-cell leukemias) showed reactivity with S-HCL 2 mAb over the full range of B-cell differentiation. In particular, very early B cells in cytoplasmic Ig (cIg)-negative, CD19-positive pre-pre-B-cell leukemias and hybrid leukemias (mixed myeloid and B-cell type) were consistently positive for CD72 on the cell surface. Therefore, CD72 may become an important marker for progenitor B-cell leukemias.     

Alkaline phosphatase-positive B cell lymphomas

Alkaline phosphatase (ALP) activity of 70 cases of non-Hodgkin's lymphomas of the B-cell type was studied. ALP activity was found in malignant lymphoma (ML), follicular, small cleaved cell (1/5 cases); ML, diffuse, small cleaved cell (3/13 cases); and mantle zone lymphoma (intermediate lymphocytic lymphoma) (2/2 cases). The ALP-positive neoplastic cells simultaneously displayed the characteristic immunophenotype of mantle zone (MZ) B lymphocytes of secondary follicle (SIg D+, BA-1+, IL-2R+ and Leu-1+). All other B-cell lymphomas, including ML, follicular, mixed small cleaved and large cell (9 cases); ML, follicular, large cell (4 cases); ML, mixed small and large cell (7 cases); ML, diffuse, large cell (27 cases); and ML, small noncleaved cell (3 cases), were consistently negative for ALP. The present study indicates that ALP-positive lymphomas including follicular lymphomas and diffuse lymphomas may be neoplastic counterparts of ALP-positive MZ B lymphocytes.     

BCL6 rearrangement in a patient with mantle cell lymphoma

 We describe a patient with mantle cell lymphoma (MCL) associated with BCL6 gene rearrangement. MCL is a distinct subtype of non-Hodgkin's lymphoma characterized by CD5+, CD10–, CD20+, t(11;14)(q13;q32) and PRAD1/cyclin D1 overexpression. Although rearrangement of the BCL6 gene is the most frequent genetic change among diffuse lymphomas and some follicular lymphomas this is the first report of a patient with MCL associated with BCL6 rearrangement. Received: 6 January 1997 / Accepted: 17 February 1997     

Costimulatory signal through CD86 is important in Th2 response in Trichinella spiralis infected mice

In order to study the role of the costimulatory signals in Th2 cytokine production, monoclonal antibodies (mAbs) against cell adhesion molecules (CAMs) were added to cultured cells obtained from the mesenteric lymph nodes (MLNs) of mice infected with Trichinella spiralis, followed by a determination of interleukin (IL)-5 and IL-4 in the culture supernatant. IL-5 production by MLN cells stimulated with somatic antigen was significantly reduced by addition of anti-CD86 but not by anti-CD80 mAb. Combination of anti-CD80 and anti-CD86 mAbs reduced IL-5 production most effectively. IL-4 production induced by anti-CD3 mAb was suppressed only by the addition of anti-CD86 mAb. Blockade of the ICAM-1/LFA-1 and VCAM-1/VLA-4 interactions was less effective on the production of IL-5 and IL-4 than the addition of anti-CD86 mAb alone. In contrast to the in vitro cytokine production, intraperitoneal injection of anti-CD80, anti-CD86 mAb, or both, similarly suppressed the peak of the eosinophilia on day 21. Elevation of somatic antigen-specific IgE and IgG1 levels as well as total IgE was not inhibited by the administration of anti-CD80, anti-CD86 mAb or both. In-vitro and in-vivo effects of CTLA-4 immunoglobulin were similar to those of combined treatment with anti-CD80 and anti-CD86 mAbs. These results suggest that the interaction between antigen-presenting cells and CD4 T cells through CD86 are most important in Th2 response during T. spiralis infection.     

Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiency with normal or elevated IgM.

B lymphocytes from patients with X chromosome-linked immunoglobulin deficiency with normal or elevated serum IgM are unable to switch from the synthesis of IgM/IgD to that of other immunoglobulin isotypes. Isotype switch recombination was evaluated in three affected males by examining interleukin 4-driven IgE synthesis. T-cell-dependent IgE synthesis was completely absent in the B lymphocytes of the patients. In contrast, CD40 mAb plus interleukin 4 induced the patients' B cells to synthesize IgE and to undergo deletional switch recombination. Because interaction between CD40 and its ligand on activated T cells is critical for T-cell-driven isotype switching, we examined CD40 ligand expression. In contrast to normal T cells, lymphocytes from the patients expressed no detectable CD40 ligand on their surface after stimulation with phorbol 12-myristate 13-acetate and ionomycin, although the mRNA of the ligand was expressed normally. These results suggest that defective expression of the CD40 ligand underlies the failure of isotype switching in this disease.     

Intracellular IL-4/IFN-gamma producing peripheral T lymphocyte subsets in B cell non-Hodgkin's lymphoma patients

AIM: The availability of several biological therapy options is rapidly growing in the field of malignant lymphomas. This emphasizes the need to understand the precise interaction of the host immune system with the malignant disease. We measured the intracellular cytokine responses in lymphoma patients' lymphocytes, to characterize the polarization changes in their immune system. METHODS: Patients with B cell non-Hodgkin's lymphoma were involved in the study. Peripheral lymphocytes were labeled with anti-CD4 or anti-CD8 antibodies and intracellular accumulation of IL-4 or IFN-gamma was detected after in vitro incubation the cells with activating cocktail. The frequency of different T-cell subsets were measured within the lymphocyte population. RESULTS: Significantly increased CD4+, IFN-gamma producing (Th1) cell percentage were found in untreated lymphoma cases (28.8% vs. 21.8%). CD8+ IL-4 and IFN-gamma producing (Tc0) T cell frequency is significantly higher in untreated lymphoma patients compared with normal controls (1.3% vs. 0.47%). The frequency of CD4+ IL-4 producing (Th2) cells is significantly lower in untreated patients (0.96% vs. 1.19%). Patients in long-term remission have lower frequency of CD4+, IL4 producing (Th2) cell ratio (0.31% vs. 1.19%) and increased CD4+ IFN-gamma producing (Th1) cell frequency (30.1% vs. 21.8%), compared with healthy normal controls. CONCLUSION: Our results demonstrate that there is a sustained increase in the CD4+ IFN-gamma producing cell frequency in lymphoma patients. The frequency of CD4+ IL-4 producing cells is decreasing by treatment. These may contribute to strong polarization toward Th1 type response, needed for lymphoma clearance and remission.     

BLyS and BLyS receptor expression in non-Hodgkin's lymphoma

OBJECTIVE: B Lymphocyte Stimulator (BLyS) protein and its receptor are new members of the tumor necrosis factor family, with specific effects exclusively on B cells. We have studied the tumor cell expression of the BLyS-Receptor (BLyS-R) and the serum BLyS protein levels in patients with different types of non-Hodgkin's lymphomas (NHL). METHODS: BLyS-R expression was assessed by flow cytometry on B cells from 43 NHL patients and 10 normal donors. BLyS protein serum levels were analyzed by ELISA. RESULTS: All B cells, tumor and normal, expressed BLyS-R. The mean fluorescence intensity (MFI +/- SD) of BLyS-R on normal B cells was 25.2 +/- 2.3 arbitrary units, while follicular NHL and chronic lymphocytic leukemia (CLL) exhibited significantly lower expression of the BLyS-R (17.7 +/- 3.1; 15.5 +/- 3.9, respectively, p < 0.0001 for both); other lymphoma subtypes expressed levels comparable to normal B cells (diffuse large cell, 24.8 +/- 4.3; mantle cell, 20 +/- 4.7; marginal zone, 20.7 +/- 3.7). BLyS protein serum levels were analyzed in 15 normal donors and 17 patients with follicular NHL. Levels of BLyS protein were, on average, threefold higher in patients with follicular lymphoma compared to normal donors (mean +/- SD; 13.4 +/- 5.6 ng/mL vs 4.6 +/- 0.7 ng/mL; p < 0.0001). BLyS protein alone was unable to stimulate proliferation in cultures of follicular lymphoma B cells or normal B cells. CONCLUSION: The specificity of the expression of BLyS-R by B-cell lymphomas opens new opportunities for the treatment of these cancers by targeting this ligand-receptor pair.     

Platelet Derived Endothelial Cell Growth Factor/Thymidine Phosphorylase Enhanced Human IgE Production

BackgroundAngiogenesis is one pathogenesis of allergic airway disease.Methodspotent angiogenic factor is platelet-derived endothelial cell growth factor (PD-ECGF), also known as thymidine phosphorylase (TP) in the field of cancer-associated research. Vascular endothelial growth factor (VEGF) is another representative angiogenic factor. Both factors were added to the culture system of human peripheral blood mononuclear cells (PBMC) with IL-4 and anti-CD40 monoclonal antibody (mAb). Total IgE levels in the supernatants and signal transduction of stimulated PBMC were evaluated.ResultsAddition of PD-ECGF enhances in vitro IgE production by PBMC in the presence of IL-4 and anti- CD40 mAb, but VEGF does not enhance IgE production. Although PD-ECGF catalyzes the reversible phosphorolysis of thymidine to 2-deoxy-D-ribose-1-phosphate (2DDR), treatment of 2DDR has no effect on IgE production by human PBMC. Both IL-4 and anti-CD40 mAb induce PD-ECGF by human PBMC. Thymidine phosphorylase inhibitor (TPI), 5-chloro-6-[1- (2-iminopyrrolidinyl) methyl] uracil hydrochloride reduce IgE production via blocking of STAT6- phosphorylation.ConclusionsTaken together, these results suggest TP involvement in the enhancement of IgE production and suggest that TPI is a novel strategy against IgE-related allergic disease.     

CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies

Antibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells after activation, including NK cells. In the present study, we show that an anti-CD137 agonistic mAb enhances the antilymphoma activity of rituximab by enhancing ADCC. Human NK cells up-regulate CD137 after encountering rituximab-coated tumor B cells, and subsequent stimulation of these NK cells with anti-CD137 mAb enhances rituximab-dependent cytotoxicity against the lymphoma cells. In a syngeneic murine lymphoma model and in a xenotransplanted human lymphoma model, sequential administration of anti-CD20 mAb followed by anti-CD137 mAb had potent antilymphoma activity in vivo. These results support a novel, sequential antibody approach against B-cell malignancies by targeting first the tumor and then the host immune system.     

Expression of interleukin-2 receptor beta subunit in hematopoietic malignancies

The expression of the interleukin-2 (IL-2) receptor was studied in neoplastic cells derived from acute leukemias, T-cell lymphoblastic lymphomas, peripheral T-cell lymphomas, chronic lymphocytic leukemias, well-differentiated lymphocytic lymphomas, and established cell lines by both flow cytometric analysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) after affinity crosslinking of radiolabeled IL-2. Cells from most acute leukemias (19 of 22), irrespective of their subtype (T, common or nonlymphoid leukemias), as well as T-cell lymphoblastic lymphomas and peripheral T- cell lymphomas expressed only the p70-75 beta subunit of the IL-2 receptor. Cells from the more mature B-cell neoplasms, chronic lymphocytic leukemia, and well-differentiated lymphocytic lymphoma, expressed predominantly alpha beta IL-2 receptors (11 of 14). In contrast to these results, most cell lines established from hematopoietic malignancies do not express either chain of the IL-2 receptor. Further studies are necessary to determine the exact function of the IL-2R p70-75 beta subunit in immature hematopoietic cells, but its wide distribution throughout the hematopoietic system suggests that IL-2 may play a role in the early stages of hematopoiesis.     

The expression of CD26 and CD40 ligand is mutually exclusive in human T- cell non-Hodgkin's lymphomas/leukemias

CD26 and CD40 ligand (CD40L) are surface molecules on human activated T lymphocytes that play a critical role in the regulation of lymphopoiesis. Both molecules are expressed on a restricted fraction of human T-cell non-Hodgkin's lymphomas (NHL)/leukemias; however, little is known about their functional and/or clinical significance in these disorders. In this study, the pattern of expression of CD40L was compared with that of the CD26 molecule. A series of 67 human T-cell NHL/leukemias and a panel of leukemia/lymphoma T-cell lines were evaluated by immunohistochemistry, flow cytometry, and RNA studies. The overall frequency of CD26+ and CD40L+ samples was rather similar (25/67 [37%] v 18/67 [27%]). However, the majority of CD26-expressing cases clustered in the lymphoblastic lymphomas (LBL)/T-acute lymphoblastic leukemias (ALL; 12/23) and CD30+ anaplastic large-cell (ALC) lymphomas (5/8), whereas CD40L+ lymphomas included a large fraction of mycosis fungoides (11/21 [52%]). CD26 and CD40L coexpression was found only in 2 myocosis fungoides cases and 1 small lymphocytic lymphoma. Thus, the expression of the two antigens was mutually exclusive in almost all T- cell lymphomas/leukemias. Accordingly, lymphoma cell lines expressed either one of the molecules or the relative amounts of CD26 and CD40L were inversely proportional. In contrast, reactive T lymphocytes from patients with non-neoplastic T-cell expansions and in vitro activated CD3+ or CD4+ normal T cells were found to coexpress CD40L and CD26. Results of a multivariate analysis showed that the expression of CD26 in T-cell LBL/ALL patients was associated to a worse outcome in terms of survival, as compared with patients with CD26- tumors (P < or = .0001). Based on our results, it can be concluded that, (1) as opposed to activated or reactive normal T cells, the expression of CD26 and of CD40L is mutually exclusive in human T-cell lymphomas/leukemias; (2) expression of CD26 is restricted to aggressive pathologic entities, such as T-cell LBL/ALL and T-cell CD30+ ALC lymphomas, whereas CD40L is expressed on slow progressing diseases such as mycosis fungoides; and (3) within the T-cell LBL/ALL group of tumors, CD26 may identify a subset of poor prognosis patients.     

Expression of the human OX40 (hOX40) antigen in normal and neoplastic tissues

Summary. The Ber-ACT35 mAb was raised against the human T-cell lymphotrophic virus (HTLV) 1-transformed, CD4⁺ HUT 102 cell line and recognizes the human homologue of the 0X40 (hOX40) antigen. The analysis of the expression of hOX40 by immunohistochernical techniques in malignant lymphomas, carcinomas and non-malignant tissues of different organs shows that hOX40 expression was almost completely restricted to T lymphocytes. Besides T cells only a small subpopulation of macrophages in Langerhans'cell histiocytosis and a few blasts in B-cell non-Hodgkin's lymphoma (B-NHL) revealed a faint immunostaining with the Ber-ACT35 mAb. Furthermore, most of the hOX40⁺ T-cells are CD4⁺.