Abstract
CD56bri natural killer (NK) cells play an important role in the pathogenesis of graft-vs. -host disease (GVHD) and immune defense in the early period after allogeneic hematopoietic stem cell transplantation. Extracorporeal photopheresis (ECP) as an immunomodulating therapy has been widely used for GVHD treatment. However, the mechanism of action of ECP still remains to be elucidated, particularly the influence of ECP on NK cells. Thirty-four patients with steroid-refractory/resistant acute GVHD (aGVHD) ≥ °II and moderate to severe chronic GVHD (cGVHD) received ECP therapy. Patient samples obtained during intensive and long-term treatment were analyzed. Immunomonitoring with respect to cell phenotype and function was performed on rested peripheral blood mononuclear cells (PBMCs) using multiparametric flow cytometry. NK activity in terms of cytokine release was analyzed by intracellular cytokine staining after co-culture with K562 cells. Moreover, the proliferative capacity of NK cells, CD4+, and CD8+ T cells was determined by carboxyfluorescein succinimidyl ester (CFSE) staining. Clinically, 75% of aGVHD and 78% of cGVHD patients responded to ECP therapy. Moreover, our data show that aGVHD, cGVHD patients and healthy donors (HDs) present distinct NK patterns: aGVHD patients have a higher frequency of CD56bri NK subsets with stronger NKG2D and CD62L expression, while CD56−CD16+ NK cells with higher expression of CD57 and CD11b stand out as a signature population for cGVHD. ECP therapy could significantly decrease CD56briCD16− NK cells with shifting the quality from a cytotoxic to a regulatory pattern and additionally mature CD56dim NK cells via upregulation of CD57 in complete responding aGVHD patients. Moreover, ECP could keep the anti-viral and anti-leukemic effects intact via maintaining specialized anti-viral/leukemic CD57+NKG2C+CD56dim NK cells as well as remaining the quality and quantity of cytokine release by NK cells. The proliferative capacity of effector cells remained constant over ECP therapy. In conclusion, ECP represents an attractive option to treat GVHD without compromising anti-viral/leukemic effects. Shaping of CD56bri NK cell compartment by downregulating the cytotoxic subset while upregulating the regulatory subset contributes to the mechanisms of ECP therapy in aGVHD.
Highlights
Extracorporeal photopheresis (ECP) is being widely used for the treatment of T cell-mediated diseases e.g., graft-vs.-host disease (GVHD) with established clinical benefits [1]
Patients suffering from steroid-refractory/resistant acute GVHD (aGVHD) were characterized by a higher frequency of CD56bri natural killer (NK) cells when compared with healthy donors (HDs) and patients with chronic GVHD (cGVHD) (Figure 1C)
Our results show that a significant decrease of cytotoxic CD27−CD11b+ NK cells was observed in aGVHD patients after ECP therapy (Supplementary Figure 2A), caused by the dramatic reduction of cytotoxic NK cells within the CD56bri NK cell populations (Supplementary Figure 2B, Figures 5A,B)
Summary
Extracorporeal photopheresis (ECP) is being widely used for the treatment of T cell-mediated diseases e.g., graft-vs.-host disease (GVHD) with established clinical benefits [1]. Clinical data illustrated that a low frequency of CD56bri NK cells is associated with the development of GVHD [12, 13]. 5 × 105 rested PBMCs were co-cultured with 5 × 104 K562 cells in the presence of CD107a antibody at 37◦C, 5% CO2 in a 96U-bottom plate for 6 hours. Surface Marker Staining After 10 min blocking at 4◦C, 5 × 105 rested PBMCs were stained with different antibodies for 20 min at 4◦C in the dark. Thereafter, cells were stained with surface marker antibodies, followed by fixation and permeabilization according to the Miltenyi Foxp fix/perm buffer instruction. One-way ANOVA with Bonferroni post-hoc test was performed to assess the differences of the marker expression and the cytokine release pattern among HDs, patients with aGVHD and cGVHD within the five different NK subsets. A p-value < 0.05 was considered to be statistically significant
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