BACKGROUND: CD56 bright natural killer (NK) regulatory cells were recently shown to display a differential impact on the risk of developing extensive chronic graft-versus-host disease (GVHD). To date no study has definitively established which immune populations are most responsible for the immunomodulatory effects or response to extracorporeal photopheresis (ECP) for GVHD. STUDY DESIGN AND METHODS:To test the role of CD56 bright NK cells in ECP, a prospective enhanced flow cytometry follow-up of immune subsets (CD19+, CD3+, CD3+/CD4+, CD3+/CD8+, CD3-/CD56+, CD3-/ CD56 bright , and CD3-/CD56 dim ) was performed in 32 patients with GVHD who underwent 552 procedures. RESULTS: An early increase of CD56 bright NK cells wasfound as a hallmark effect to ECP, particularly during the first 3 months of treatment. This was also supported by the ability to predict for complete responses when this increase was expressed as a higher CD56 bright versus CD56 dim NK cells ratio. Among the immune subsets tested, the only variable that had direct influence on response to ECP was a CD56 bright/dim ratio more than 0.16 (hazard ratio [HR] 4.32, p = 0.014; HR 5.8, p = 0.007, at 2 and 3 months of ECP treatment, respectively). CONCLUSION:These findings argue for exploring strategies for priming a CD56 bright NK cell expansion during ECP and providing additional and potentially relevant data for revisiting the underpinning cellular mechanisms of ECP that could generate that expansion.ABBREVIATIONS: aGVHD = acute graft-versus-host disease; alloHSCT = allogeneic hematopoietic stem cell transplantation; cGVHD = chronic graft-versus-host disease; CR = clinical response; ECP = extracorporeal photopheresis; IQR = interquartile range. From theFig. 3. CD56 bright/dim NK ratio as predictor of response to ECP in GVHD. ROC features for the ratio achieved after (A) 2 months and (B) 3 months of ECP treatment. Kaplan-Meier curves for cumulative probability of achieving complete response to ECP for a CD56 bright/dim NK ratio > 0.16 after (C) 2 months and (D) 3 months. AUC = area under the curve. [Color figure can be viewed at wileyonlinelibrary.com] Volume 58, December 2018 TRANSFUSION 2929 CD56 BRIGHT NK CELLS AND ECP

Graft versus host disease (GvHD) is the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we report studies of a patient with chronic GvHD (cGvHD) carrying persistent CD4+ T cell clonal expansion harboring somatic mTOR, NFKB2, and TLR2 mutations. In the screening cohort (n = 134), we detect the mTOR P2229R kinase domain mutation in two additional cGvHD patients, but not in healthy or HSCT patients without cGvHD. Functional analyses of the mTOR mutation indicate a gain-of-function alteration and activation of both mTORC1 and mTORC2 signaling pathways, leading to increased cell proliferation and decreased apoptosis. Single-cell RNA sequencing and real-time impedance measurements support increased cytotoxicity of mutated CD4+ T cells. High throughput drug-sensitivity testing suggests that mutations induce resistance to mTOR inhibitors, but increase sensitivity for HSP90 inhibitors. Our findings imply that somatic mutations may contribute to aberrant T cell proliferations and persistent immune activation in cGvHD, thereby paving the way for targeted therapies.

Peripheral expansion of cytotoxic T lymphocytes (CTL) derived from the graft in the initial stages of allogeneic haematopoietic stem cell transplantation (alloHSCT) immune recovery is a well-known physiological event. The description of symptomatic large granular lymphocyte leukaemia in this setting may generate uncertainty, mostly in those cases in which the CTL expansion (CTLe) persists beyond the early transplantation period. We aimed to assess the nature of CTLe during the post-alloHSCT period in 154 adult patients with a long-term surveillance. We studied the longitudinal kinetics of those expansions, their relationship to clinical events, and their phenotypic and molecular features, including recently reported CTL leukaemia-STAT3 mutations. Persistent relative CTLe cases are frequent (49%), related with thymoglobulin prophylaxis (P ≤ 0·001), acute graft-versus-host disease (GVHD, P = 0·02), and reduced intensity conditioning (P = 0·04). Absolute CTLe are scarce (9%) and related to chronic GVHD. T cell receptor rearrangement was reported as clonal and oligoclonal in the majority of patients with CTLe. The absence of STAT3 mutations and the CD8/CD4 declining longitudinal kinetics in the late period supports its benign nature, expressed clinically by the null detrimental impact of these expansions on post-transplant outcome and/or serious infectious events.