In Vitro PUVA Treatment Preferentially Induces Apoptosis in Alloactivated T Cells : Transplantation

Journal Logo

Letters to the Editor

In Vitro PUVA Treatment Preferentially Induces Apoptosis in Alloactivated T Cells

Holtick, Udo1,2; Wang, Xiao N.3; Marshall, Scott R.3; von Bergwelt-Baildon, Michael1,2; Scheid, Christof2; Dickinson, Anne M.3

Author Information
doi: 10.1097/TP.0b013e31825f4454
  • Free

Extracorporeal photopheresis (ECP) has been demonstrated to reduce graft-versus-host disease (GvHD) after allogeneic hematopoietic transplantation without causing generalized immunosuppression (1). One hypothesis to explain this observation is a higher susceptibility of alloactivated T cells to ECP-induced apoptosis. The ability of ECP to induce apoptosis in lymphoid cells was first reported by Marks and Fox (2) and further scrutinized by Bladon and Taylor (3, 4). Since then, it has been speculated that preferential killing of activated lymphocytes could be one reason for the selective effects of ECP, but only a few studies have been published. Heng et al. (5) observed a larger number of apoptotic cells after psoralen plus ultraviolet (PUVA) treatment of phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) as compared with unstimulated PBMCs, but their data failed to reach statistical significance. More recently, Hannani et al. (6) reported in this journal that human leukocyte antigen DR–positive T cells derived from three patients with chronic GvHD were significantly more sensitive to ECP than human leukocyte antigen DR–negative T cells. Similarly, OKT3 and PHA-activated T cells were more prone to undergo apoptosis after in vitro PUVA treatment as compared with unstimulated T cells. Here, we have tested the hypothesis, using single cell sorting and flow cytometry of alloactivated T cells, that recognition of alloantigens may also lead to enhanced susceptibility to ECP-induced apoptosis.

Irradiated healthy donor PBMCs were used as stimulators for third party responder PBMCs in three independent experiments. Proliferating allogeneic mixed lymphocyte reaction (MLR) cells were harvested after 72 hr of culture. The cells were left untreated or incubated with 8-methoxypsoralen and ultraviolet A treated (in vitro PUVA) to mimic ECP treatment as previously described (7). T cells were then analyzed by flow cytometry for markers of activation (CD25/CD69) and apoptosis (Annexin V/PI). The percentage of live cells was lower in CD69-positive and CD25-positive cells as compared with the CD69-negative and CD25-negative untreated cells, but this difference was not significant. Twenty-four hours after PUVA treatment, the median percentage of live cells as defined by Annexin V and PI negativity was decreased in CD25/CD69 double-positive cells (29% vs. 54%, P=0.04, Fig. 1A), CD25-positive T cells (36% vs. 51%, Fig. 1C), and CD69-positive cells (29% vs. 56%, P=0.041, Fig. 1E). A median of 63% apoptosis was induced in the CD25 CD69 double-positive cells as compared with 40% in the double-negative subset (Fig. 1B). In CD25-positive and CD69-positive cells, a median of 52% (vs. 44%) and 60% (vs. 41%) apoptosis was induced (Fig. 1D,F).

A–F, Multicolor flow cytometry of day +3 mixed lymphocyte reaction cells 24 hr after in vitro PUVA treatment. Percentage of live cells (A) and difference in apoptosis induction (B) in CD25+ CD69+ vs. CD25 CD69 T cells. Percentage of live cells (C) and difference in apoptosis induction (D) in CD25+ vs. CD25 T cells. Percentage of live cells (E) and difference in apoptosis induction (F) in CD69+ cells vs. CD69 T cells (* P<0.05, Mann-Whitney U test). G and H, Analysis of fluorescence-activated cell sorter–sorted T cells. After 72 hr of mixed lymphocyte reaction culture, cells were harvested and stained for CD3, CD25, and CD69. T cells were sorted by fluorescence-activated cell sorter sorting and in vitro PUVA treated or left untreated. G, Apoptosis induction in sorted CD25 CD69 double-positive T cells was compared with CD25 CD69 double-negative cells. H, Comparison of the percentage of late apoptotic cells (Annexin V and PI double positive) (* P<0.05, Mann-Whitney U test).

To confirm these observations, CD69/CD25 double-positive T cells were sorted by flow cytometry in a separate set of MLR experiments and then treated or untreated with in vitro PUVA. Results were compared with sorted T cells double-negative for CD25 and CD69 also treated or untreated with PUVA. Susceptibility to PUVA-induced apoptosis was significantly enhanced in the CD25 CD69 double-positive T cells. The median percentage of live cells 24 hr after PUVA-treatment was 41.35% in double negative cells and 18.46% in double positive cells (P=0.0407, Fig. 1G). The percentage of late apoptotic cells was 27.24% and 58.01%, respectively (Fig. 1H).

The results obtained in this study reveal a significantly higher susceptibility of alloactivated T cells to PUVA-induced apoptosis. Moreover, we demonstrate by cell sorting that this susceptibility is independent of MLR bystander cell influence. The data suggest selective killing of activated cells; however, PUVA also induces apoptosis in nonactivated T cells. Activation-induced cell death could synergize with PUVA efficacy, but untreated activated T cells had only slightly higher background apoptosis than nonactivated T cells. Ultraviolet A–induced DNA intercalation of 8-methoxypsoralen might also have higher impact on highly activated and proliferating T cells.

In vivo, predominant killing of activated T cell clones can cause anti-idiotype responses against these clones. It has been demonstrated that T-cell receptors of clonal origin can become targets of T-cell receptor–specific anti-idiotype immune responses (8, 9). In CTCL, circulating clonally amplified malignant T cells are reduced by ECP treatment (10).

So far, there is little evidence to support this hypothesis in the setting of GvHD, although oligoclonal T-cell populations have also been described in autoimmune diseases and chronic GvHD (11). French et al. (12) furthermore reported improved response rates to ECP treatment in patients with GvHD when oligoclonal T cells were present in the peripheral blood. Data from animal autoimmune and transplantation models imply that the process of photopheresis could induce specific effects against clonally amplified T-cell populations (13–15).

Our results demonstrate that preferential killing of alloactivated T cells may contribute to the mode of action of ECP/PUVA treatment. Whether anti-idiotype vaccination responses are involved warrants further research.

Udo Holtick


Xiao N. Wang3

Scott R. Marshall3

Christof Scheid2

Michael von Bergwelt-Baildon1,2

Anne M. Dickinson3

1Cologne Interventional Immunology University Hospital of Cologne Cologne, Germany

2 Stem Cell Transplantation Program Department I of Internal Medicine University Hospital of Cologne Cologne, Germany

3 Haematological Sciences Institute of Cellular Medicine Newcastle University Newcastle upon Tyne, UK


1. Marshall SR. Technology insight: ECP for the treatment of GvHD—can we offer selective immune control without generalized immunosuppression? Nat Clin Pract Oncol0 2006; 3: 302.
2. Marks DI, Fox RM. Mechanisms of photochemotherapy-induced apoptotic cell death in lymphoid cells. Biochem Cell Biol 1991; 69: 754.
3. Bladon J, Taylor PC. Extracorporeal photopheresis induces apoptosis in the lymphocytes of cutaneous T-cell lymphoma and graft-versus-host disease patients. Br J Haematol 1999; 107: 707.
4. Bladon J, Taylor PC. Lymphocytes treated by extracorporeal photopheresis demonstrate a drop in the Bcl-2/Bax ratio: a possible mechanism involved in extracorporeal-photopheresis–induced apoptosis. Dermatology 2002; 204: 104.
5. Heng AE, Sauvezie B, Genestier L, et al.. PUVA apoptotic response in activated and resting human lymphocytes. Transfus Apher Sci 2003; 28: 43.
6. Hannani D, Merlin E, Gabert F, et al.. Photochemotherapy induces a faster apoptosis of alloreactive activated T cells than of nonalloreactive resting T cells in graft versus host disease. Transplantation 2010; 90: 1232.
7. Holtick U, Marshall SR, Wang XN, et al.. Impact of psoralen/UVA-treatment on sur-vival, activation, and immunostimulatory capacity of monocyte-derived dendritic cells. Transplantation 2008; 85: 757.
8. Offner H, Vainiene M, Gold DP, et al.. Protection against experimental encephalomyelitis. Idiotypic autoregulation induced by a nonencephalitogenic T cell clone expressing a cross-reactive T cell receptor V gene. J Immunol 1991; 146: 4165.
9. Zhang L, Jayne DR, Oliveira DB. Anti-idiotype antibodies to anti-mitochondrial antibodies in the sera of patients with primary biliary cirrhosis. J Autoimmun 1993; 6: 93.
10. Rook AH. Photopheresis in the treatment of autoimmune disease: experience with pemphigus vulgaris and systemic sclerosis. Ann N Y Acad Sci 1991; 636: 209.
11. French LE, Rook AH. T cell clonality and the effect of photopheresis in systemic sclerosis and graft versus host disease. Trans-fus Apher Sci 2002; 26: 191.
12. French LE, Alcindor T, Shapiro M, et al.. Identification of amplified clonal T cell populations in the blood of patients with chronic graft-versus-host disease: positive correlation with response to photopheresis. Bone Marrow Transplant 2002; 30: 509.
13. Berger C, Perez M, Laroche L, et al.. Inhibition of autoimmune disease in a murine model of systemic lupus erythematosus induced by exposure to photoinactivated lymphocytes. J Invest Dermatol 1990; 94: 52.
14. Girardi M, Herreid P, Tigelaar RE. Specific suppression of lupus-like graft-versus-host disease using extracorporeal photochemical attenuation of effector lymphocytes. J Invest Dermatol 1995; 104: 177.
15. Gatza E, Rogers CE, Clouthier SG, et al.. Extracorporeal photopheresis reverses experimental graft-versus-host disease through regulatory T cells. Blood 2008; 112: 1515.
© 2012 Lippincott Williams & Wilkins, Inc.