Malignant B Cells Induce the Conversion of CD4+CD25− T Cells to Regulatory T Cells in B-Cell Non-Hodgkin Lymphoma

Recent evidence has demonstrated that regulatory T cells (Treg) were enriched in the tumor sites of patients with B-cell non-Hodgkin lymphoma (NHL). However, the causes of enrichment and suppressive mechanisms need to be further elucidated. Here we demonstrated that CD4+CD25+FoxP3+CD127lo Treg were markedly increased and their phenotypes were different in peripheral blood (PB) as well as bone marrow (BM) from newly diagnosed patients with B-cell NHL compared with those from healthy volunteers (HVs). Involved lymphatic tissues also showed higher frequencies of Treg than benign lymph nodes. Moreover, the frequencies of Treg were significantly higher in involved lymphatic tissues than those from PB as well as BM in the same patients. Suppression mediated by CD4+CD25+ Treg co-cultured with allogeneic CFSE-labeled CD4+CD25− responder cells was also higher in involved lymphatic tissues from B-cell NHL than that mediated by Treg from HVs. In addition, we found that malignant B cells significantly induced FoxP3 expression and regulatory function in CD4+CD25− T cells in vitro. In contrast, normal B cells could not induce the conversion of CD4+CD25− T cells to Treg. We also showed that the PD-1/B7-H1 pathway might play an important role in Treg induction. Taken together, our results suggest that malignant B cells induce the conversion of CD4+CD25− T cells to Treg, which may play a role in the pathogenesis of B-cell NHL and represent a promising therapeutic target

Pretreatment platelet count predicts survival outcome of patients with de novo non-M3 acute myeloid leukemia

Background Pretreatment platelet count has been reported as a potential tool to predict survival outcome in several solid tumors. However, the predictive value of pretreatment platelet count remains obscure in de novo acute myeloid leukemia (AML) excluding acute promyelocytic leukemia (M3). Methods We conducted a retrospective review of 209 patients with de novo non-M3 AML in our institute over a period of 8 years (2007–2015). Receiver operating characteristic (ROC) curve analysis was used to determine the optimal platelet (PLT) cutoff in patients. We analyzed the overall survival (OS) and disease free survival (DFS) using the log-rank test and Cox regression analysis. Results By defining the platelet count 50 × 109/L and 120 × 109/L as two cut-off points, we categorized the patients into three groups: low (120 × 109/L). On univariate analysis, patients with medium platelet count had longer OS and DFS than those with low or high platelet count. However, the multivariate analysis showed that only longer DFS was observed in patients with medium platelet count than those with low or high platelet count. Conclusion Our findings indicate that pretreatment platelet count has a predictive value for the prognosis of patients with non-M3 AML

MOESM1 of Increased Th17 cells and IL-17A exist in patients with B cell acute lymphoblastic leukemia and promote proliferation and resistance to daunorubicin through activation of Akt signaling

Additional file 1: Table S1. The sequences of the primers used for real-time qPCR

Elevated frequencies of CD4⁺CD25⁺FoxP3⁺CD127^lo Treg in PB, BM and involved lymphatic tissues in patients with B-cell NHL.

<p>(A) Dot plots were gated on CD4⁺ T cells (based on forward and side scatter and CD4 staining). The number in the dot plot represents the percentage of gated cells expressing the relevant marker. (B) Frequencies of CD4⁺CD25⁺FoxP3⁺CD127^lo Treg in PB, BM and involved lymphatic tissues from patients with B-cell NHL and HVs were quantified using flow cytometric analysis. (C) Frequencies of CD4⁺CD25⁺FoxP3⁺CD127^lo Treg in PB, BM and involved lymphatic tissues from the same patients with B-cell NHL were measured as described earlier. (D) Frequencies of CD4⁺CD25⁺FoxP3⁺CD127^lo Treg in PB, BM and involved lymphatic tissues from indolent B-cell NHL patients and aggressive B-cell NHL patients were measured as described earlier. (E) Frequencies of CD4⁺CD25⁺FoxP3⁺CD127^lo Treg in BM and involved lymphatic tissues from patients with B-cell NHL were measured as described earlier. Each open circle represents a single individual assessed in the respective group and numbers on the left of horizontal bars represent the group means.</p

Functional analysis of induced FoxP3-expressing CD4 cells in vitro.

<p>(A) CD4⁺CD25⁻ cells isolated from a patient with B-cell NHL were co-cultured with purified autologous B cells for 5 days. Then, CD4⁺CD25⁺ T cells were isolated and cocultured with CFSE-labeled CD4⁺CD25⁻ T cells for 4 days in the presence of γ-irradiated (30 Gy) PBMCs, with each population 2×10⁵ cells. Histograms showed the profile of CFSE-labeled CD4⁺CD25⁻ T cells and the proliferated CD4⁺CD25⁻ T cells was measured by the percentage of CFSE^dim cells, as indicated. The converted Treg could inhibit the proliferation of allogeneic CD4⁺CD25⁻ T cells (<i>P</i><0.01). Two BM samples (patients 8 and 11) and one LN sample (patient 24) were tested, and a representative experiment of three was presented. (B) CD4⁺CD25⁻ cells isolated from a HV or patient with B-cell NHL were co-cultured with purified respective autologous B cells for 5 days. Then, the cells were stimulated with PMA plus ionomycin for 5 hours in the presence of BFA. Cells were harvested and costained with anti-CD3 and anti-CD8. Next, cells were fixed, permeabilized and stained with anti-FoxP3, anti-IL-2, anti-IFN-γ, anti-IL-4, or anti-IL-17. Dot plots were gated on CD3⁺CD4⁺(CD3⁺CD8⁻) T cells, and the numbers within each quadrant represent the percentages of CD4⁺ T cells. Two BM samples (patients 8 and 11) and one LN sample (patient 24) were tested, and the results were representatives of three independent experiments.</p

Clinical characteristics of patients with B-cell NHL.

<p>DLBCL indicates diffuse large B-cell lymphoma; MCL, mantle cell lymphoma; new, a diagnosis of lymphoma with no previous history of lymphoproliferative disease; MZL, marginal zone lymphoma; and SLL, small lymphocytic lymphoma.</p

Malignant B cells induce FoxP3 expression and Treg conversion from CD4⁺CD25⁻ T cells, which partly depends on the interaction of between PD-1 and B7-H1.

<p>(A,B) Malignant B cells, but not normal B cells, were able to induce FoxP3 expression in autologous conventional T cells. CD4⁺CD25⁻ T cells were cultured for 5 days with X-ViVO™ 15 medium alone or purified autologous B cells in the presence of 100 IU/ml IL-2. The cells were then harvested, stained, and analyzed for the percentage of CD4⁺FoxP3⁺ cells. The number in the dot plot represents the percentage of gated cells expressing the relevant marker. Each open circle represents a single individual and numbers on the left of horizontal bars represent the group means. The results were representative of seven independent experiments. (C,D) Malignant B cells, but not tumor-infiltrating T cells, were able to induce the conversion of Treg from conventional T cells. CD4⁺CD25⁻ T cells isolated from involved lymphatic tissues of B-cell NHL were co-cultured with normal B cells for 5 days. CD4⁺CD25⁻ T cells isolated from PB of HVs were co-cultured with malignant B cells for 5 days. (E) The expression of B7-H1 was higher in lymphoma B cells than normal B cells. Cells were costained with anti-CD19-FITC and anti-B7-H1-PE, and then were analyzed the expression of B7-H1 on CD19⁺ B cells using flow cytometry. (F,G) Histograms showed the effect of the interaction between PD-1 and B7-H1 on the conversion of Treg from conventional T cells. CD4⁺CD25⁻ T cells were cocultured with autologous B cells purified from patients with B-cell NHL for 5 days. Cells were treated with PD-1 fusion protein or anti-B7-H1 antibody as well as their corresponding controls in the coculture system. A CD4⁺FoxP3⁺ gate was drawn based on their isotype controls. The results were representative of seven independent experiments.</p