B-lymphocyte stimulator (BLyS) stimulates immunoglobulin production and malignant B-cell growth in Waldenström macroglobulinemia

Waldenström macroglobulinemia (WM) is a serious and frequently fatal B-cell malignancy associated with an elevated monoclonal IgM protein in the serum. Many of the mechanisms leading to this disease are not yet known. B-lymphocyte stimulator (BLyS) is a TNF family member that is critical for maintenance of normal B-cell development and homeostasis. BLyS is overexpressed in a variety of B-cell malignancies and has been shown to inhibit apoptosis in malignant B cells. It also regulates immunoglobulin secretion by normal B cells. To determine the relevance of BLyS in WM, we examined the role of BLyS in WM patient samples. Malignant B cells were found to bind soluble BLyS and variably express the receptors BAFF-R, TACI, and BCMA. We also found expression of BLyS in bone marrow specimens by immunohistochemistry and elevated serum BLyS levels in patients with WM. BLyS, alone or in combination with cytokines that induce immunoglobulin production, was found to increase IgM secretion by malignant B cells. Furthermore, BLyS was found to increase the viability and proliferation of malignant B cells from WM patients. Due to the role of BLyS in WM, strategies to inhibit BLyS may potentially have therapeutic efficacy in these patients

Soluble and Membrane-Bound TGF-β-Mediated Regulation of Intratumoral T Cell Differentiation and Function in B-Cell Non-Hodgkin Lymphoma

<div><p>While the effect of TGF-β on malignant B cells in non-Hodgkin lymphoma (NHL) has been previously evaluated, studies to specifically define the role of TGF-β in tumor immunity in B-cell NHL are limited. We found that soluble TGF-β, secreted by both lymphoma cells and intratumoral T cells, is present in the serum of patients with B-cell NHL. Soluble TGF-β promoted regulatory T (T_reg) cells by enhancing expression of Foxp3 in CD4⁺ T cells and suppressed effector helper T (T_H) cells by inhibiting expression of IFN-γ and IL-17. Blockade of the IL-2 signaling pathway diminished the effect of soluble TGF-β on T cell differentiation. Furthermore, we found that membrane-bound TGF-β is expressed specifically on the surface of malignant B cells in B-cell NHL. TGF-β was able to bind to the surface of lymphoma B cells through an interaction with heparan sulfate (HS) but not through the TGF-β receptor. We showed that pretreatment of lymphoma B cells with TGF-β significantly inhibits the proliferation and cytokine production of intratumoral T cells. Taken together, these results suggest that tumor-associated soluble and membrane-bound TGF-β are involved in the regulation of intratumoral T cell differentiation and function in B-cell NHL.</p> </div

Effects of TGF-β on IL-2 production in T cells.

<p>(A) Representative dot plots showing the expression of IL-2 in CD4⁺ T cells treated with or without TGF-β. Data acquired from multiple samples were summarized (right, n = 8). (B) IL-2 levels measured by ELISA in culture supernatants of freshly-isolated intratumoral T cells cultured in anti-CD3-coated plate with escalating doses of TGF-β (n = 3). R: resting T cells. (C) Representative dot plots (n = 3) showing the expression of IL-2 in CD4⁺ T cells treated with or without IL-12 or TGF-β alone or in combination. (D) Representative dot plots (n = 5) showing the expression of IL-2 in CD4⁺ CD45RA⁺ naïve or CD4⁺CD45RO⁺ memory T cells treated with or without TGF-β in the presence of IL-1β plus IL-6.</p

Effects of IL-2 signaling on TGF-β-mediated regulation of T cell differentiation in B-cell NHL.

<p>(A) Representative dot plots (n = 6) showing the expression of IFN-γ and IL-17 in CD4⁺ T cells treated with or without αIL-2 or αIL-2Rα or β in the presence or absence of TGF-β plus IL-6 and IL-23. (B) Representative dot plots (n = 3) showing the expression of Foxp3 and CD25 in CD4⁺ T cells treated with or without TGF-β, αIL-2 or αIL-2Rα or β alone or in combination. (C) Dot plots from a representative sample (n = 5) showing the expression of IFN-γ, IL-17 and IL-2 in CD4⁺ T cells treated with or without TGF-β or αIL-2.</p

Effect of TGF-β on the differentiation of intratumoral T cells in B-cell NHL.

<p>(A) Representative histograms (n = 3) showing proliferation measured by CFSE staining of CD4⁺ or CD8⁺ T cells treated with different doses of TGF-β. Proliferative capacity was expressed by calculating the number of CFSE^dim cells. (B) Representative dot plots showing the expression of Foxp3, IFN-γ or IL-17 in CD4⁺, CD4⁺CD45RA⁺ or CD4⁺CD45RO⁺ T cells treated with or without TGF-β. (C) Summary of the numbers of T_reg (CD4⁺Foxp3⁺), T_H1 (CD4⁺IFN-γ⁺) or T_H17 (CD4⁺IL-17⁺) cells induced by TGF-β. (D) Representative dot plots (n = 3) showing the expression of IFN-γ in CD4⁺ T cells treated with or without TGF-β or IL-12 or IL-23 alone or in combination. (E) Representative dot plots (n = 10) showing the expression of IL-17 in CD4⁺ T cells treated with or without IL-1β plus IL-6. (F) Representative dot plots (n = 6) showing the expression of IL-17 in CD4⁺ T cells treated with or without TGF-β in the presence of IL-1β plus IL-6.</p

Soluble TGF-β expression in B-cell NHL.

<p>(A) TGF-β serum levels measured by ELISA in untreated FL patients (mean: 27.67+/−13.2, n = 10) and healthy donors (median: 30.76+/−9.47 pg/ml, n = 10; p = 0.56). (B) TGF-β levels in culture supernatants of freshly-isolated malignant B (B, n = 11) and intratumoral T cells (T, n = 5) from B-cell NHL treated with (A) or without (R) PMA/Ion measured by ELISA. (C) A representative graph showing TGF-β levels in culture supernatants of B and T cell lines treated with (Stim) or without (Unstim) PMA/Ion measured by ELISA (n = 3).</p