Seventeen of the 23 patients in this cohort were included in our previous series.17 Patient and donor demographics and details of conditioning are in Table 1. tumor cells to stimulate allogeneic T-cell immune responses which may have wider consequences for adoptive immunotherapy strategies. Introduction Follicular lymphoma (FL) is usually a common germinal center B-cell malignancy characterized by slow progression but inevitable relapse after conventional chemoimmunotherapy.1,2 FTY720 (Fingolimod) However, some patients can be cured by the graft-versus-lymphoma (GVL) effect provided by donor T cells in the setting of allogeneic hematopoietic stem cell transplantation (AHSCT).3 FL B cells carry the hallmark t(14;18) translocation which results in cytoplasmic overexpression of the Bcl-2 protein. Two recent studies have reported that additional tumor-specific genetic aberrations of the tumor necrosis factor receptor superfamily 14 (aberrations on clinical outcome, suggesting that their functional effects might be influenced by factors such as differing treatment approaches.4,5 HVEM is a type I transmembrane molecule which acts as a molecular switch through interactions with several different ligands including B- and T-lymphocyte attenuator (BTLA), LIGHT, CD160, lymphotoxin A, and glycoprotein D to regulate a range of immune responses.6 Conversation between HVEM expressed on antigen-presenting cells and the coinhibitory receptor BTLA on T cells limits T-cell activation and proliferation.7 BTLA has intracellular immunoreceptor tyrosine-based inhibition motifs consistent with immune-inhibitory function, and BTLA-deficient animal models display exaggerated immune responses.6 Importantly, BTLA is expressed by naive CD4+ and CD8+ T cells, the T-cell compartments known to be enriched for alloreactive specificity, and agonistic antibody-mediated BTLA stimulation reduces donor T-cellCmediated acute GVHD in murine transplant models, consistent with a functional role for BTLA in controlling donor T-cell alloresponses in this setting.8-10 Activated FL B cells can act as potent alloantigen-presenting cells in vitro11 and patients with FL often undergo FTY720 (Fingolimod) AHSCT with significant residual lymphoma. We hypothesized that aberrations would reduce expression of HVEM and increase the ability of FL B cells to stimulate allogeneic T-cell responses. We therefore decided the functional effect of aberrations around the alloantigen-presenting capacity of human FL B cells in vitro. We also decided the impact of aberrations on clinical alloreactivity in FL patients after HLA-matched reduced-intensity conditioning AHSCT. Materials and methods Patient samples Lymph node biopsies FTY720 (Fingolimod) were obtained from FL patients after written consent. The study was approved by the Local Research Ethical Committee (05/Q0605/140) and was conducted in accordance with the Declaration of Helsinki. mutation and deletion analysis of FL B cells Tumor DNA from pre-AHSCT lymph node biopsies from FL patients was screened for mutations by polymerase chain reaction amplification/Sanger sequencing and for deletions by multiplex ligation-probe amplification as previously described.12 Primers used for Sanger sequencing are summarized in supplemental Table 1 (available on the Web site). FL B-cell sorting, activation, and phenotyping FL B FTY720 (Fingolimod) cells were stained with CD10Cfluorescein isothiocyanate (clone 97C5) and CD20Cperidinin chlorophyll (PerCP; clone LT20) antibodies (both from Miltenyi Biotec) and purified by fluorescence-activated cell sorting of dual-positive events on a FACSAria device (Becton Dickinson). Dead cells were excluded using 4,6-diamidino-2-phenylindole (DAPI). Purity of sorted FL B cells was routinely >90% and sorted FL B cells were routinely >95% light chainCrestricted assessed with anti-immunoglobulin light chain CAlexa Fluor 700 (clone MHK-49) and anti-immunoglobulin light chain Callophycocyanin (APC; clone MHL-38) antibodies (supplemental Physique 1). Following sorting, FL B cells were activated for 48 hours with 1 g/mL soluble CD40L (InVivoGen), 5 g/mL AffiniPure F(ab)2 fragment goat anti-human immunoglobulin A (IgA) + IgG + IgM (H+L; Jackson ImmunoResearch), 5 g/mL CpG (R&D SERPINF1 Systems), and 50 ng/mL interleukin-4 (IL-4; R&D Systems) to optimally upregulate expression of molecules involved in antigen presentation as previously described.13,14 Immunophenotyping of CD10+CD20+ FL B cells was performed by flow cytometry using the following antibodies: HVEM-phycoerythrin (PE; clone 122), CD58-PE (clone TS2/9), major histocompatibility complex (MHC) class ICPacific Blue (clone W6/32) HLA-DRCAPC (clone L243), CD80-PE-cyanine 7 (Cy7; 2D10), CD86-APC (clone IT2.2), and their corresponding isotype controls (all from Biolegend). Measurement of FL-B-cellCstimulated T-cell alloresponses Untouched CD3+ T cells were purified by unfavorable selection from.