CD56bright natural killer regulatory cells in filgrastim primed donor blood or marrow products regulate chronic graft- versus-host disease: the Canadian Blood and Marrow Transplant Group randomized 0601 study results

Abstract

Randomized trials have conclusively shown higher rates of chronic graft-versus-host disease with filgrastim-stimulated apheresis peripheral blood as a donor source than unstimulated bone marrow. The Canadian Blood and Marrow Transplant Group conducted a phase 3 study of adults who received either filgrastim-stimulated apheresis peripheral blood or filgrastim-stimulated bone marrow from human leukocyte antigen-identical sibling donors. Because all donors received the identical filgrastim dosing schedule, this study allowed for a controlled evaluation of the impact of stem cell source on development of chronic graft-versus-host disease. One hundred and twenty-one evaluable filgrastim-stimulated apheresis peripheral blood and filgrastim-stimulated bone marrow patient donor products were immunologically characterized by flow cytometry and tested for their association with acute and chronic graft-versus-host disease within 2 years of transplantation. The immune populations evaluated included, regulatory T cells, central memory and effector T cells, interferon γ positive producing T cells, invariate natural killer T cells, regulatory natural killer cells, dendritic cell populations, macrophages, and activated B cells and memory B cells. When both filgrastim-stimulated apheresis peripheral blood and filgrastim-stimulated bone marrow were grouped together, a higher chronic graft-versus-host disease frequency was associated with lower proportions of CD56bright natural killer regulatory cells and interferon γ-producing T helper cells in the donor product. Lower CD56bright natural killer regulatory cells displayed differential impacts on the development of extensive chronic graft-versus-host disease between filgrastim-stimulated apheresis peripheral blood and filgrastim-stimulated bone marrow. In summary, while controlling for the potential impact of filgrastim on marrow, our studies demonstrated that CD56bright natural killer regulatory cells had a much stronger impact on filgrastim-stimulated apheresis peripheral blood than on filgrastim-stimulated bone marrow. This supports the conclusion that a lower proportion of CD56bright natural killer regulatory cells results in the high rate of chronic graft-versus-host disease seen in filgrastim-stimulated apheresis peripheral blood. clinicaltrials.gov Identifier: 00438958.

Trial registration: ClinicalTrials.gov NCT00438958.

Copyright© Ferrata Storti Foundation.

Figures

Figure 1.

Algorithm of sample analysis for immune population that correlate with the development of overall cGvHD. G-PB: G peripheral blood; cGvHD: chronic graft-versus-host disease; aGvHD: acute graft-versus-host disease; IFN: interferon; NK: natural killer; DC: dendritic cells; TRM: transplant related mortality.

Figure 2.

Correlation of donor CD56bright NKreg cells infusion characteristics with acute and chronic GvHD. (A) Box and whisker plot of CD56bright NKreg cells percentage per total lymphocytes for overall cGvHD, which was calculated as the total nucleated cells gated on FSC/SSC plots, then the percentage of each subpopulation of interest was determined as a percentage of the total number of lymphocytes (based on forward and side scatter). The phenotype of CD56bright NK cells was CD56bright CD3-CD16-perforin-granzyme B. (B) The correlation of CD56bright NKreg cells with extensive cGvHD. (C) Representative dot plot showing CD56bright and CD56dim subpopulations of NKreg cells. Mononuclear cells were stained by appropriate conjugated mAbs for surface markers. The cells were then fixed and treated to be permeable, followed by intracellular staining (Online Supplementary Table S1). The data was acquired by LSRII equipped with four lasers. A minimum of 1×10 cells were acquired. Single cells were gated based on FSC-A vs. FSC-H and dead cells were excluded using fixable viability dye. Lymphocytes were gated as FSC lo and SSC lo. Upper left dot plot: NK cells were defined as CD3−CD56+ cells. Two populations are revealed based on expression of CD56, CD56bright (gate A) and CD56dim (gate B). CD56brightcells express a significantly higher level of activating receptor CD335 (NKp46) compared to CD56dim. CD56bright cells express a significantly lower level of molecules involved in killing, Granzyme B (bottom left dot plot) and Perforin (bottom right dot plot). (D) A ROC analysis was used to determine an ‘optimal’ cut point for correctly predicting the occurrence of overall chronic GvHD. ‘Optimal’ was defined as the point on the ROC curve with the shortest distance from the point (0, 1). The point (0, 1) represents the ideal, 100% sensitivity and 100% specificity. GvHD: graft-versus-host disease; NK: natural killer; AUC: area under the curve.

Figure 3.

Correlation of donor IFNγ producing CD4+ T cells infusion characteristics with acute and chronic GvHD. (A) Box and whisker plot of the correlation of IFNγ producing CD4+ T cells percentage with overall cGvHD. The population was calculated as a subpopulation of CD3+ lymphocytes. PBMCs were stimulated with PMA (100ng/ml)/Ionomycin (1 μg/ml) in the presence of monensin for 6 hours. Unstimulated cells treated with monensin were used as control. At the end of incubation, the cells were harvested and surface markers were stained for CD3, CD4, and CD8, in addition to fixable viability to distinguish dead cells. Intracellular staining was performed to detect production of IL-17, IL-4 and IFNγ. The data was acquired using BD LSRII and analyzed by FlowJo v9. Hierarchical gating; 1: lymphocytes were selected based on FSC/SSC, 2: exclusion of dead cells, 3: selection of CD3+ T cells, and 4: determination of IFNγ producing CD4+ T-cells. The data is presented as % of CD4+IFNγ+ T cells per CD3+ lymphocytes. (B) Correlation of IFNγ producing CD4+ T cells with extensive cGvHD; (C) A representative dot plot of INFγ+ T helper cells. Mononuclear cells were seeded at a density of 1×10 per milliliter of culture medium (RPMI-1640 supplemented with 10% heat-inactivated FBS and 2mM l-glutamine), and the cells incubated in CO2 incubator providing 95% oxygen and 5% CO2 at 37 degrees centigrade. The cells were stimulated for 6 hours with 100ng/ml PMA and 1 μg/ml Ionomycin in presence of golgi inhibitor monensin (BD Biosciences; following manufacturers’ instructions). As control, cells were cultured without stimulator, but received monensin. The cells were then harvested and surface staining was performed to detect CD3+CD4+ cells. Then the cells were fixed and treated to be permeable. Intracellular staining was performed to detect IFNγ, IL-4 and IL-17. At least 1×10 cells were acquired. Gating hierarchy; 1: single cells (FSC-A vs. FSC-H), 2: viable cells (fixable viability dye negative), and 3: lymphocytes 4-CD3+CD4+ cells. The percentage of Th1 (IFNγ+), Th17 (IL-17+) and Th2 (IL-4+) were determined after setting quadrant based on unstimulated cells (upper row). Dot plots in lower row show cytokine expression after stimulation. D) A ROC analysis was used to determine an ‘optimal’ cut point for correctly predicting the occurrence of chronic GvHD. The optimal cut point for IFNγ producing CD4+ T cells/CD3+ T cells was 13.9%. (E) Logistic regression was performed with the two identified markers as predictors and overall cGvHD status as response. Each sample was given an estimated probability of overall cGvHD based on the fitted model. The ROC was generated by applying different probability thresholds. GvHD: graft-versus-host disease; IFN: interferon; AUC: area under the curve.

Figure 4.

Impact of donor IFNγ producing CD4+ T cells and CD56bright NKreg cells infusion characteristics on cGvHD by donor source using G-PB or G-BM transplantation. (A) The estimated probability of overall cGvHD by treatment (donor source G-BM versus G-PB) as a function of the CD56bright cells per total lymphocytes. B) Estimated probability of extensive cGvHD as a function of CD56bright cells per total lymphocytes by donor source (G-PB or G-BM). C) Estimated probability of overall cGvHD as a function of donor IFNγ producing CD4+ T cells by donor source (G-PB or G-BM); D) Estimated probability of extensive cGvHD as a function of donor IFNγ producing CD4+ T cells by donor source (G-PB or G-BM). GvHD: graft-versus-host disease; IFN: interferon.