In vitro effects of different 8-methoxypsoralen treatment protocols for extracorporeal photopheresis on mononuclear cells

Holger Budde, Ulrike Berntsch, Joachim Riggert, Tobias J Legler, Holger Budde, Ulrike Berntsch, Joachim Riggert, Tobias J Legler

Abstract

Extracorporeal photopheresis (ECP) is an important second-line therapy for graft-versus-host disease. A central therapeutic mechanism is the induction of immune tolerance through apoptosis in patient's leukocytes, caused by ex vivo incubation with 8-methoxypsoralen (8-MOP) and subsequent UVA irradiation. We hypothesized that different 8-MOP incubation times and an additional 8-MOP removal step could influence the apoptosis kinetics of leukocytes in general and in particular could lead to different apoptotic levels in the leukocyte subpopulations. After 8-MOP/UVA treatment of human leukocytes, cells were cultured and the percentage of annexin V positive cells from several leukocyte subpopulations was determined. Only regulatory T cells (Tregs) were relatively resistant to 8-MOP/UVA induced apoptosis. When cells were incubated for 30 minutes with 8-MOP prior to UVA exposure, higher percentages of annexin V positive cells were detected on day 1 and day 2 after treatment. Removal of 8-MOP after UVA exposure caused no significant changes in the apoptosis kinetics during the 72 h culture period compared with unwashed cells. The results of our in vitro study indicate that it could be possible to adjust the apoptosis kinetics via modulation of the 8-MOP incubation time. In further in vivo studies it should be elucidated to which extent different apoptosis kinetics influence the therapeutic effect of ECP since steady-state apoptosis levels are probably important for establishing a long lasting immune tolerance. Furthermore we found that Tregs, according to their well-known tolerogenic function, are more resistant to apoptosis after 8-MOP/UVA treatment compared to GvHD inducing T cell populations.

Keywords: apoptosis; photopheresis; regulatory T cells (Tregs).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Apoptosis of leukocytes and monocytes after different 8-MOP/UVA treatments. Leukocytes were stained for the pan leukocyte marker CD45 and for annexin V as apoptosis marker and analyzed by flow cytometry (A). Monocytes were detected by CD14 staining (B). Percentages of annexin V positive cells out of all CD45 or CD14 cells are shown in the bar chart. Treatment conditions for Figs. 1-3: Without treatment (ctrl.), with 30 minutes’ 8-MOP pre-incubation and subsequent washing step for 8-MOP removal before culturing (30 min pre-incubation + wash), with 8-MOP addition immediately before UVA irradiation and subsequent 8-MOP removal before culturing (no pre-incubation + wash) and with 8-MOP addition immediately before UVA irradiation without 8-MOP removal before culturing (no pre-incubation w/o wash). Apoptosis of untreated ctrl. samples after 1, 2 and 3 days in culture was always significantly lower (p < 0.001) compared with treated samples (Figs. 1-3). Other significant differences are depicted with * p < 0.05, ** p < 0.01 or *** p < 0.001
Fig. 2
Fig. 2
Apoptosis of T cells after different 8-MOP/UVA treatments. Leukocytes were stained with CD3 as pan T cell marker (A), CD4 as T helper cell marker (B), CD8 as cytotoxic T cell marker (C) and with CD4 CD25 co-staining as regulatory T cell marker (D). Percentages of annexin V positive cells out of all cells from the corresponding cell type are shown in the bar charts. For treatment conditions, see legend of Fig. 1
Fig. 3
Fig. 3
Apoptosis of B cells, NK cells and NKT cells after different 8-MOP/UVA treatments. B cells were identified with marker CD19 (A). NK cells were defined as CD56 positive and CD3 negative cells (B) and NKT cells as CD56 CD3 double-positive cells (C). Percentages of annexin V positive cells out of all cells from the corresponding cell type are shown in the bar charts. For treatment conditions, see legend of Fig. 1

References

    1. Blazar BR, Murphy WJ, Abedi M. Advances in graft-versus-host disease biology and therapy. Nat Rev Immunol. 2012;12:443–458.
    1. Bluestone JA, Auchincloss H, Nepom GT, et al. The Immune Tolerance Network at 10 years: tolerance research at the bedside. Nat Rev Immunol. 2010;10:797–803.
    1. Merlin E, Paillard C, Rochette E, et al. Extracorporeal photochemotherapy as second- or first-line therapy of acute GVHD? Bone Marrow Transplant. 2010;45:963–965.
    1. Marshall SR. Technology insight: ECP for the treatment of GvHD-can we offer selective immune control without generalized immunosuppression? Nat Clin Pract Oncol. 2006;3:302–314.
    1. Suchin KR, Cassin M, Washko R, et al. Extracorporeal photochemotherapy does not suppress T- or B-cell responses to novel or recall antigens. J Am Acad Dermatol. 1999;41:980–986.
    1. Wolf CE, Meyer M, Riggert J. Leukapheresis for the extraction of monocytes and various lymphocyte subpopulations from peripheral blood: product quality and prediction of the yield using different harvest procedures. Vox Sang. 2005;88:249–255.
    1. Heshmati F. Mechanisms of action of extracorporeal photochemotherapy. Transfus Apher Sci. 2003;29:61–70.
    1. Voss CY, Fry TJ, Coppes MJ, et al. Extending the horizon for cell-based immunotherapy by understanding the mechanisms of action of photopheresis. Transfus Med Rev. 2010;24:22–32.
    1. Durazzo TS, Tigelaar RE, Filler R, et al. Induction of monocyte-to-dendritic cell maturation by extracorporeal photochemotherapy: initiation via direct platelet signaling. Transfus Apher Sci. 2014;50:370–378.
    1. Lamioni A, Parisi F, Isacchi G, et al. The immunological effects of extracorporeal photopheresis unraveled: induction of tolerogenic dendritic cells in vitro and regulatory T cells in vivo. Transplantation. 2005;79:846–850.
    1. Jiang H, Lu Z, Pan S, et al. Opposite effects of donor apoptotic versus necrotic splenocytes on splenic allograft tolerance. J Surg Res. 2006;136:247–254.
    1. Grodzicky T, Elkon KB. Apoptosis: a case where too much or too little can lead to autoimmunity. Mt Sinai J Med. 2002;69:208–219.
    1. Hackstein H, Amoros JJ, Bein G, et al. Successful use of miniphotopheresis for the treatment of graft-versus-host disease. Transfusion. 2014;54:2022–2027.
    1. Birge RB, Ucker DS. Innate apoptotic immunity: the calming touch of death. Cell Death Differ. 2008;15:1096–1102.
    1. Alcindor T, Gorgun G, Miller KB, et al. Immunomodulatory effects of extracorporeal photochemotherapy in patients with extensive chronic graft-versus-host disease. Blood. 2001;98:1622–1625.
    1. Perfetti P, Carlier P, Strada P, et al. Extracorporeal photopheresis for the treatment of steroid refractory acute GVHD. Bone Marrow Transplant. 2008;42:609–617.
    1. Porto G, Giordano RJ, Marti LC, et al. Identification of novel immunoregulatory molecules in human thymic regulatory CD4+CD25+ T cells by phage display. PLoS One. 2011;6:e21702.
    1. Chen X, Subleski JJ, Hamano R, et al. Co-expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood. Eur J Immunol. 2010;40:1099–1106.
    1. Ward DM. Extracorporeal photopheresis: how, when, and why. J Clin Apher. 2011;26:276–285.
    1. Gatza E, Rogers CE, Clouthier SG, et al. Extracorporeal photopheresis reverses experimental graft-versus-host disease through regulatory T cells. Blood. 2008;112:1515–1521.
    1. Florek M, Sega EI, Leveson-Gower DB, et al. Autologous apoptotic cells preceding transplantation enhance survival in lethal murine graft-versus-host models. Blood. 2014;124:1832–1842.
    1. Budde H, Kolb S, Salinas Tejedor L, et al. Modified extracorporeal photopheresis with cells from a healthy donor for acute graft-versus-host disease in a mouse model. PLoS One. 2014;9:e105896.
    1. Maeda A, Schwarz A, Bullinger A, et al. Experimental extracorporeal photopheresis inhibits the sensitization and effector phases of contact hypersensitivity via two mechanisms: generation of IL-10 and induction of regulatory T cells. J Immunol. 2008;181:5956–5962.
    1. Apostolou A, Williams RE, Comereski CR. Acute toxicity of micronized 8-Methoxypsoralen in rodents. Drug Chem Toxicol. 1979;2:309–313.
    1. Dubertret L, Averbeck D, Zajdela F, Bisagni E, Moustacchi E, Touraine R, Latarjet R. Photochemotherapy (PUVA) of psoriasis using 3-carbethoxypsoralen, a non-carcinogenic compound in mice. Br J Dermatol. 1979;101:379–389.
    1. Gasparro FP, Dall’Amico R, Goldminz D, et al. Molecular aspects of extracorporeal photochemotherapy. Yale J Biol Med. 1989;62:579–593.
    1. Bladon J, Taylor PC. Extracorporeal photopheresis: a focus on apoptosis and cytokines. J Dermatol Sci. 2006;43:85–94.
    1. Karolak L, Tod M, Leon A, et al. In vitro kinetics of 8-methoxypsoralen penetration into human lymphoid cells. Photodermatol Photoimmunol Photomed. 1992;9:58–60.
    1. Roos WP, Kaina B. DNA damage-induced cell death by apoptosis. Trends Mol Med. 2006;12:440–450.
    1. Schmid D, Grabmer C, Streif D, et al. T-cell death, phosphatidylserine exposure and reduced proliferation rate to validate extracorporeal photochemotherapy. Vox Sang. 2015;108:82–88.
    1. Xia CQ, Campbell KA, Clare-Salzler MJ. Extracorporeal photopheresis-induced immune tolerance: a focus on modulation of antigen-presenting cells and induction of regulatory T cells by apoptotic cells. Curr Opin Organ Transplant. 2009;14:338–343.
    1. Lorenz K, Rommel K, Mani J, et al. Modulation of lymphocyte subpopulations by extracorporeal photopheresis in patients with acute graft-versus-host disease or graft rejection. Leuk Lymphoma. 2015;56:671–675.
    1. Di Biaso, Di Maio L, Bugarin C, et al. Regulatory T cells and extracorporeal photochemotherapy: correlation with clinical response and decreased frequency of proinflammatory T cells. Transplantation. 2009;87:1422–1425.
    1. Hannani D, Gabert F, Laurin D, et al. Photochemotherapy induces the apoptosis of monocytes without impairing their function. Transplantation. 2010;89:492–499.
    1. Tambur AR, Ortegel JW, Morales A, et al. Extracorporeal photopheresis induces lymphocyte but not monocyte apoptosis. Transplant Proc. 2000;32:747–748.
    1. Cutler C, Kim HT, Bindra B, et al. Rituximab prophylaxis prevents corticosteroid-requiring chronic GVHD after allogeneic peripheral blood stem cell transplantation: results of a phase 2 trial. Blood. 2013;122:1510–1517.
    1. Alousi AM, Uberti J, Ratanatharathorn V. The role of B cell depleting therapy in graft versus host disease after allogeneic hematopoietic cell transplant. Leuk Lymphoma. 2010;51:376–389.
    1. Palmer JM, Rajasekaran K, Thakar MS, et al. Clinical relevance of natural killer cells following hematopoietic stem cell transplantation. J Cancer. 2013;4:25–35.

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