Short treatment of peripheral blood cells product with Fas ligand using closed automated cell processing system significantly reduces immune cell reactivity of the graft in vitro and in vivo

Abstract

Mobilized peripheral blood cells (MPBCs) graft and peripheral blood cells apheresis are used for bone marrow transplantation and for treatment of graft versus host disease (GvHD). We demonstrate that a short treatment of MPBCs with Fas ligand (FasL, CD95L) for 2 h using a closed automated cell processing system selectively induces apoptosis of specific donor T cells, B cells and antigen presenting cells, but, critically, not CD34⁺ hematopoietic stem cells and progenitors, all of which may contribute to an increased likelihood of graft survival and functionality and reduced GvHD. Treated cells secreted lower levels of interferon-gamma as compared with control, untreated, cells. Moreover, FasL treatment of immune cells increased signals, which led to their phagocytosis by activated macrophages. FasL treated immune cells also reduced the ability of activated macrophages to secrete pro-inflammatory cytokines. Most importantly, FasL ex vivo treated MPBCs prior to transplantation in NOD-SCID NSG mice prevented GvHD and improved stem cell transplantation in vivo. In conclusion, MPBCs, as well as other blood cell products, treated with FasL by automated manufacturing (AM), may be used as potential treatments for conditions where the immune system is over-responding to both self and non-self-antigens.

Fig. 1. FasL AM MPBCs treatment significantly reduces CD3⁺ cells without compromising the quantity and quality of CD34⁺ cells.

a FasL AM MPBCs treatment process performed using the Fresenius Kabi LOVO functionally closed automated cell processing system, designed for HSCT. MPBCs graft characterization following (b–f) 100 ng/ml FasL AM treatment or (g–k) 400 ng/ml FasL AM treatment. Percentage of (b, g) CD3⁺ and (d, i) CD34⁺ cells per total CD45⁺ population. Percentage of annexin V positive (c, h) CD3⁺ and (e, j) CD34⁺ cells per 7AAD⁻ population. f, k Percentage of multi potent stem and progenitor cells (CD34⁺CD38^neg/low) per total CD34⁺7AAD⁻ population. b–e: N = 16, f: N = 9, g–k: N = 3. Mean ± SEM, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; Paired T-test.

Fig. 2. FasL AM MPBCs treatment does not affect the expansion capacity of CD34⁺ cells.

Characterization of HSPCs purified from 100 ng/ml FasL AM treated (a–h) or 400 ng/ml FasL AM treated (i–p) MPBCs and from control MPBCs followed by 7 days expansion. Total cell number per well, (b, j) percent of CD34⁺ cells per total cells in culture. Percentage of HSPCs subpopulations per total CD34⁺ population: (c, k) self-renewal HSCs (CD34⁺CD38^neg/low CD90⁺CD45RA⁻), (d, l) MPPs (CD34⁺CD38^neg/low CD90⁻CD45RA⁻), (e, m) LMPPs (CD38^Neg/LowCD90⁻CD45RA⁺), (f, n) OPPs (CD38^+high) (g, o) early-stage MkPs (h, p) and more mature MkPs (CD38^neg/lowCD41a⁺ and CD38^highCD41a⁺, respectively). N = 3, Mean ± SEM, *P ≤ 0.05 Paired T-test.

Fig. 3. FasL AM treatment of MPBCs decreases B cells, myeloid cells and activated T cells populations, reducing the antigen presentation potential and T cell activation potential.

MPBCs graft characterization following 100 ng/ml FasL AM treatment: (a) Percentage of B-lymphocytes (CD19⁺), (b) Myeloid cells (CD33⁺) and (c) Activated T cells (CD25⁺) per total CD45⁺ population. The percentage of HLA-DR^high expressing on B-lymphocytes (d) and on myeloid cells (e) as marker for antigen presentation potential per total CD19⁺ or CD33⁺ population. N = 7, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001; Paired T-test. f Concentration of IFN-γ secreted to the medium, measured 24 h post T cell specific and normalized per seeded CD3⁺ cell number in culture. N = 8, *P ≤ 0.05; Paired T-test.

Fig. 4. FasL AM treatment of MPBCs increases their phagocytosis and reduces proinflammatory cytokine secretion by activated macrophages.

100 ng/ml FasL AM treated and non-treated MPBCs were incubated with activated macrophages at different ratios. Percentage of high phagocytic (CFSE^+high) macrophages per total CD11b⁺ population as well as secretion of TNFα, IL-6 and IL-8 were evaluated. Percentage of high phagocytic (CFSE^+high) macrophages: (a) Representative flow cytometry dot plots, (b) results of the representative experiment are presented as mean ± SEM of technical duplicates, *P ≤ 0.05; ***P ≤ 0.001, Multiple unpaired T-test. c The effect of engulfment of apoptotic cells by activated macrophages on TNFα, IL-8 and IL-6 secretion. Results of a representative experiment are presented as Mean ± SEM of technical duplicates. Macrophages: cell ratio 1:5; *P ≤ 0.05; Multiple unpaired T-test.

Fig. 5. FasL AM treatment of MPBCs attenuates acute GvHD in a xenogeneic GvHD model, enhances the short-term engraftment of human cells and does not impair re-constitution potential of HSPC in mice 4 weeks post administration.

GvHD score (a, c) and percent survival (b, d) of two independent experiments in γ-irradiated NSG mice transplanted with either 100 ng/ml FasL AM treated or untreated MPBCs control grafts. a, b Experiment 1: MPBCs + FasL AM and MPBCs N = 8 mice/group, Vehicle N = 2 mice/group; c, d Experiment 2: MPBCs + FasL AM and MPBCs N = 10 mice/group, Vehicle N = 3 mice/group; Mean ± SEM, Multiple T tests; Kaplan–Maier survival curve, Log-rank (Mantel–Cox) test; *P ≤ 0.05, **P ≤ 0.01. e–h Engraftment of human cells in the bone marrow of treated mice, 4 weeks post-transplantation. The absolute cell numbers of (e) human CD45⁺and (f) human CD34⁺, (g) percentage of CD34⁺ per total BM cells (BM) population; MPBCs+FasL AM N = 10, MPBCs N = 6. h CFU capacity of BMC derived from mice and cultured for 2 weeks for colony growth. MPBCs + FasL AM N = 8, MPBCs N = 3. e–h Each data point represents an individual mouse, horizontal lines represent the median of each treatment group *P ≤ 0.05, Mann–Whitney test.

Fig. 6. FasL treatment of CD34⁺ HSPCs increase their stemness.

a, b FasL treatment enhances HSPCs stemness demonstrating higher F510/F580 fluorescence ratio (FR) correlated with Glutathione (GSH) level. (a) Representative flow cytometry histogram (b) results of 3 independent experiments of MPBCs treatment with 100 ng/ml of FasL; Mean ± SEM *P ≤ 0.05; Paired T-test. c–i 7 Days expansion capacity after short (5 min, 1 h and 4 h) exposure of purified CD34⁺ cells with 100 ng/ml FasL. (c) Percentage of CD34⁺ cells per total cell population, (d) total cell number per well, (e–i) percentage of HSPCs subpopulations per total CD34⁺ population: (e) Self-renewal HSCs (CD34⁺CD38^neg/lowCD90⁺CD45RA⁻), (f) MPP (CD34⁺CD38^neg/low CD90⁻CD45RA⁻), (g) LMPP (CD38^Neg/LowCD90⁻CD45RA+), (h) OPP (CD38^+high), (i) early-stage MkPs (CD38^Neg/lowCD41a⁺), (c–h): N = 3, (i) N = 2, Mean ± SEM, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 Paired T-test.