Antigen-Specific Regulatory T Cells Cells by Photopheresis Induces

The Journal of Immunology

Intravenous Infusion of Syngeneic Apoptotic Cells by Photopheresis Induces Antigen-Speciﬁc Regulatory T Cells1

Akira Maeda,* Agatha Schwarz,* Kerstin Kernebeck,* Nicole Gross,* Yoshinori Aragane,† David Peritt,‡ and Thomas Schwarz2§

The basis of extracorporeal photopheresis is the reinfusion of leukocytes previously exposed to 8-methoxypsoralen (8-MOP) and UVA radiation. It has been approved for the palliative treatment of cutaneous T cell lymphoma and has reported benefits in autoimmune diseases, transplant rejection, and graft-vs-host disease. However, the underlying mechanism of photopheresis re- mains unresolved. Because UVB radiation can cause immune tolerance via induction of regulatory T cells, we studied whether photopheresis exerts a similar effect extracorporeally. Therefore, we established a model of photopheresis using a murine model of contact hypersensitivity. Splenocytes and lymph node cells of mice that were sensitized with dinitrofluorobenzene were exposed to 8-MOP plus UVA in vitro. Intravenous injection of these cells into naive mice caused inhibition of a hapten immune response, which was lost upon depletion of CD11c؉ cells but not T cells. Mice that received untreated cells or cells exposed to UVA or 8-MOP alone were not affected. Inhibition was cell-mediated and Ag-specific as demonstrated by transfer of tolerance from the primary recipients into naive animals, which could, however, properly respond to the unrelated hapten oxazolone. Transfer activity was -lost when cells were depleted of CD4؉ or CD25؉ subpopulations. These data suggest that photopheresis exerts its immunomodu latory effects via the induction of Ag-specific regulatory T cells. The Journal of Immunology, 2005, 174: 5968–5976.

he introduction of photochemotherapy (psoralen plus point of care device withdrawing ϳ5 billion peripheral blood leu- UVA (PUVA)3), which consists of the oral ingestion of kocytes from the patient by apheresis followed by incubation of T the photosensitizer 8-methoxypsoralen (8-MOP) followed the cells with 8-MOP and UVA exposure in an extracorporeal by total body UVA (320–400 nm) exposure, has had tremendous setting. impact for the therapy of cutaneous T cell lymphoma (CTCL) (1) Because of its safety and efficacy in the treatment of CTCL, and is still widely used. The mechanism of PUVA is thought to be which is based on experience with this regimen for nearly 20 years, primarily depletion of malignant T cells from the skin (2). Early ECP has been tried in a variety of diseases that have a suspected studies in CTCL patients considered the idea of a pheresis proce- involvement of pathogenic T cells, including rejection of organ dure bringing the malignant cells outside the body for PUVA treat- transplants, graft-vs-host disease (GvHD) and autoimmune disor- by guest on October 1, 2021. Copyright 2005 Pageant Media Ltd. ment. This formed the basis for the development of extracorporeal ders (6). The efficacy and mechanism of action in CTCL has been photopheresis (ECP). Surprisingly, several of the first patients suggested to be based upon an anti-idiotypic TCR vaccination (7). treated with photopheresis went into partial or complete remission Dying tumor cells are ingested by APC and induce an Ag (mono- (3, 4). However, the beneficial effect could not be explained by a clonal TCR)-specific CTL-mediated immune response against the simple destruction of malignant cells because only a small pro- tumor. This hypothesis has recently led to a concept to improve the portion of pathogenic T cells are treated during an extracorporeal efficacy of photopheresis by incubating treated cells overnight to cycle. This led to the supposition that an immune-mediated sys- lengthen the time apoptotic CTCL cells interact with treated APC temic anti-tumor-specific activity may be involved (5). Current before reinfusion. This intriguing hypothesis, termed transimmu- ECP treatments involve a closed-loop, sterile, patient-connected,

http://classic.jimmunol.org nization (8), awaits clinical trial investigation. However, this hypothesis is a less than satisfactory explanation for the beneﬁcial effects of ECP reported in diseases other than CTCL. Although *Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, De- these diseases are extremely heterogeneous clinically, they share partment of Dermatology, University Mu¨nster, Mu¨nster, Germany; †Department of Dermatology, Kinki University School of Medicine, Osakasayama-City, Osaka, Ja- similar underlying pathophysiological mechanisms and response pan; ‡Therakos, Exton, PA 19341; and §Department of Dermatology, University Kiel, to immunosuppressive therapies. It is unlikely that ECP induces a Kiel, Germany

Downloaded from generalized immunosuppression because patients, including those Received for publication October 14, 2004. Accepted for publication February 27, 2005. undergoing long-term ECP therapy, have no reported higher risk of developing infections or malignancies (9) and respond normally to The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance both novel and recall Ag (10). More recently, it has been suggested with 18 U.S.C. Section 1734 solely to indicate this fact. that ECP may induce Ag-speciﬁc immunomodulation, possibly via 1 This work was supported by grants from the German Research Foundation (Sonder- regulatory T cells (Tr) (11). forschungsbereich 293, B9 to T.S.; SCHW1177/1-1 to A.S and T.S.) and a grant from Therakos (to T.S.). Tr comprise a heterogeneous group of T lymphocytes, which actively inhibit immune responses (12–14). They have been rec- 2 Address correspondence and reprint requests to Dr. Thomas Schwarz, Department of Dermatology, University Kiel, Schittenhelmstrasse 7, D-24105 Kiel, Germany. ognized to play an important role in the prevention of autoimmu- E-mail address: [email protected] nity, GvHD, and transplant rejection (15–17). Clinically, there is 3 Abbreviations used in this paper: PUVA, psoralen plus UVA; CHS, contact hyper- great excitement about the potential to develop methodologies that sensitivity; CTCL, cutaneous T cell lymphoma; DC, dendritic cell; DNFB, 2,4-dini- troﬂuorobenzene; ECP, extracorporeal photopheresis; GvHD, graft-vs-host disease; can harness Tr for therapeutic activity. The complexity introduced by 8-MOP, 8-methoxypsoralen; OXA, oxazolone; Tr, regulatory T cell. the varying types and activities of Tr has added an additional

challenge. Solar/UV radiation, in particular the midwave range injection. Mice were treated with ECP-treated cells via tail-vein injection (UVB, 290–320 nm), long has been recognized to exhibit the with 0.2 ml (ϳ2.5 ϫ 106 cells) of 51Cr labeled cells. The mice were then euthanized via CO asphyxiation at 24 h. Various organs (lungs, spleen, capacity to induce immunotolerance, in part, via Ag-specific Tr 2 thymus, liver, stomach, lymph nodes, kidneys, small and large intestines, (18, 19). sensitized and unsensitized ears) were harvested, placed in individual Over the last few years it has been well documented that apo- counting tubes, weighed, and counted for radioactivity using an LKB ptotic cell clearance by APC may regulate immune responses via gamma counter. Total cpm were measured, and the percent of injected cells modulation of APC (20–22). Although the majority of this work entering the organs and cell concentration were calculated. has been conducted in vitro, excellent efficacy and mechanistic studies have recently been reported in murine models of heart al- Experimental photopheresis lograft rejection (23), response to haptens (24), OVA, and LPS- Donor mice were sensitized against DNFB. Twenty-four hours later, induced lung inflammation (25). The cellular and molecular mech- spleens and regional lymph nodes were removed and single-cell suspen- anisms of this tolerance induction have been partially identified. A sions were prepared. After the washed cells were incubated with 200 ng/ml 8-MOP for 30 min in the dark, they were UVA exposed (5 J/cm2). For subset of apoptotic receptors, responsible for recognition and UVA irradiation, a UVA high-power device (Sellamed 4000; Sellas Sun- phagocytosis of apoptotic cells, also signal the APC to decrease light) was used with an emission peak at 365 nm (output 40 mW/cm2). proinflammatory cytokines, increase anti-inflammatory cytokines After washing in PBS, the cell number was adjusted to 2.5 ϫ 108/ml and and functionally inhibit the ability to stimulate effector T cells 200 ␮l of cells were injected i.v. into naive syngeneic mice. Five days later, recipients were sensitized by application of 50 ␮l of 0.5% DNFB. Recip- (21). Recent suggestions have been made that Tr may be generated ients were challenged by application of 0.3% DNFB on the left ear 5 days following APC engagement of apoptotic cells (22). It is now clear after sensitization. After another 24 h, the ear swelling response was that ingestion of apoptotic cells is not a neutral event as once measured. thought, but an active process of immune tolerance induction, the exact nature of which is unfolding quickly. Teleologically, this Cell viability process has been described as another peripheral immune tolerance Normal donor mice were used to obtain cells from spleens and regional mechanism. Autologous cells, naturally undergoing apoptosis in lymph nodes, and single cell suspensions were prepared. After washing, the periphery, are ingested by resident phagocytes and migrate to cells were incubated with 200 ng/ml 8-MOP for 30 min in the dark, fol- 2 draining lymphatics. Here they act as a constant reminder to roving lowed by exposure to UVA radiation (5 J/cm ). These cells were incubated under standard conditions at 2 ϫ 106 cells/ml in RPMI supplemented with T cells about self (26). Only in the presence of danger signals will 10% FCS. Every day for 5 days an aliquot was removed and stained with the phagocyte be altered to become immunogenic and begin the annexin V FITC and both CD3 PerCP and CD11c allophycocyanin. These appropriate immune cascade to fight pathogens (27). samples were analyzed by flow cytometry (EPICS; Coulter). Cell death is portrayed as a line graph of annexin V-positive cells in either the CD3ϩ or In the context of these studies, we were interested in investigat- ϩ ing whether ECP exhibits the capacity to induce Ag-specific Tr. CD11c populations. For this purpose, we established an in vivo model for experimental Contact hypersensitivity (CHS) photopheresis. Using this model, we demonstrated unresponsive- ness in treated mice that was transferable to naive mice, suggesting C3H/HeN mice were sensitized by painting 50 ␮l of DNFB (0.5% in ac- the involvement of a regulatory cell population. This transferable etone/olive oil, 4:1) on the shaved back on day 0. On day 5, 20 ␮l of 0.3% ϩ DNFB was applied on the left ear, the vehicle acetone/olive oil was used by guest on October 1, 2021. Copyright 2005 Pageant Media Ltd. protection is Ag specific, dose dependent, and requires CD4 and ϩ on the right ear as a control. OXA was applied at a concentration of 2% for CD25 cells. Taken together, these data provide evidence that sensitization and at 0.5% for elicitation. Ear swelling was measured in a photopheresis may exhibit the capacity to induce Ag-specific reg- blinded fashion with a spring-loaded micrometer (Mitutoyo, Kawasaki, ulatory cells. These findings may further add to the understanding Japan) 24 h after challenge. CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle- of the mode of action of ECP, provide an explanation for the long- Ϫ treated ear in sensitized animals and was expressed in centimeters ϫ 10 3 lived efficacy and safety of ECP in the treatment of a variety of (mean Ϯ SD). Mice that were ear-challenged without prior sensitization diseases, and open up the possibility of better clinical use and served as negative controls. Each group consisted of at least seven mice. future improvements in this therapy. Each experiment was performed at least twice. http://classic.jimmunol.org Materials and Methods Adoptive cell transfer Animals and reagents Spleens and regional lymph nodes were removed from mice that had received 8-MOP/UVA-treated splenocytes (1° recipients). The cell number C3H/HeN mice (8–10 wk of age) were purchased from Harlan Winkel- was adjusted to 2.5 ϫ 108/ml and cells were injected i.v. (200 ␮l) into mann. BALB/c female mice (4–6 wk of age) were purchased from Charles naive syngeneic mice (2° recipients). Twenty-four hours later, 2° recipients River Laboratories. Animals were housed under specific pathogen-free were sensitized against DNFB. Five days later, ear challenge was per- conditions, and animal care was provided by expert personnel in compli- formed and ear swelling was measured 24 h thereafter. The scheme of ance with the relevant laws and institutional guidelines. 2,4-Dinitrofluoro- Downloaded from experimental photopheresis, including the transfer studies, is demonstrated benzene (DNFB) and oxazolone (OXA) were purchased from Sigma-Al- in Fig. 1. drich. UVADEX (8-MOP) was provided from Therakos, a Johnson & Johnson company (Exton, PA). Depletion of subpopulations Trafficking of photopheresis-treated cells For depletion of subpopulations, lymphocytes obtained from regional BALB/c mice (n ϭ 6) were sensitized on day Ϫ5 by topical application of lymph nodes and spleens were incubated with microbeads coated with Ab 20 ␮l of a 1.6% (w/v) solution of OXA in ethanol on both the inside and against CD4, CD8, CD25, CD11c, or Thy1.2 (Miltenyi Biotec). The incu- outside of the left ear. On day 0, animals were challenged with 20 ␮lof bation was followed by negative selection via magnetic bead separation 0.8% solution of OXA on the inside and outside of the left ear while the using the autoMACS system (Miltenyi Biotec). The negative fractions right ear was treated with vehicle. were harvested, washed, and adjusted to the appropriate cell numbers and On day 0, whole splenocytes from syngeneic littermate mice were subsequently used for injection. The efficacy of depletion or isolation was pooled, resuspended in medium, and radiolabeled using standard labora- determined by flow cytometric analysis (EPICS; Coulter). tory procedures. Cells were labeled with 51Cr (100 ␮Ci 51Cr per ϳ2 ϫ 107 o 6 cells) for1hat37 C, washed twice with HBSS, resuspended at 12.5 ϫ 10 Statistical analysis cells/ml, and placed in a T-75 flask. To this flask, 200 ng/ml 8-MOP was ;p Ͻ 0.005 ,ءء .added before UVA irradiation (3 J/cm2). Cells were quickly removed from All statistics were analyzed by single tail Student’s t test .p Ͻ 0.01 ,ء the flask to avoid adherence and placed at the appropriate concentration for 5970 PHOTOPHERESIS INDUCES Tr CELLS

FIGURE 1. Scheme of experimental in vivo model for CHS and ECP. Spleen and lymph node cells were obtained from mice that were sensitized with DNFB. Cells were exposed extracorporeally to 8-MOP plus UVA radiation. Treated cells were injected i.v. into naive syngeneic recipients (1° recipients). Five days after injection, recipients were sensitized against DNFB and ear challenge performed 5 days later. In some experiments, spleen and lymph node cells were obtained from the recipient mice and transferred i.v. into a second generation of syngeneic naive mice (2° recipients). Twenty-four hours after transfer, mice were sensitized against DNFB and ear challenge performed 5 days later. FIGURE 2. 8-MOP/UVA induces apoptotic cell death in virtually all cells. Single-cell suspensions of spleens and lymph nodes were incubated with 200 ng/ml 8-MOP for 30 min in the dark, followed by exposure to Results UVA radiation (5 J/cm2). Every day for 5 days, an aliquot was removed 8-MOP/UVA induces apoptosis and stained with annexin V FITC and both CD3 PerCP and CD11c APC. Splenocytes from control mice were incubated with 8-MOP and A, Linear graph of the percent total (squares), CD3 (diamonds), and CD11c exposed to UVA radiation and incubated under standard condi- (circles) which are annexin V positive on days 0–5. Untreated cells are in open symbols. B, A subset of histograms of treated cells on day 0, 1, 3, and tions for up to 5 days. These cells were analyzed by ﬂow cytometry 5 forward/side scatter and CD11c/annexin V graphs. for forward scatter and side scatter and dual stained for annexin V, CD11c, and CD3. 8-MOP/UVA-induced apoptosis of virtually all cells, including the CD3ϩ and CD11cϩ subsets. Interestingly, the bination with 5 J/cm2 UVA light and injected i.v. into naive syn- CD11cϩ cells had a slightly slower kinetic of annexin V positivity geneic mice. These recipients were sensitized with DNFB, and ear as compared with CD3ϩ cells (Fig. 2). We have also noted a sim- challenge was performed 5 days later. Ear swelling was measured

by guest on October 1, 2021. Copyright 2005 Pageant Media Ltd. ilar delay in human cell death following ECP (data not shown). 24 h after challenge (Fig. 1). Recipients of 8-MOP/UVA-treated cells had a signiﬁcantly decreased ear swelling response compared Extracorporeally 8-MOP/UVA-treated cells are retained with controls (Fig. 3). In contrast, when cells were exposed to primarily in the spleen and liver either 8-MOP (Fig. 3A) or UVA alone (Fig. 3B), ear swelling of Splenocytes from syngeneic littermate control mice were labeled the recipients was not affected. In addition, no suppression of sen- with 51Cr and exposed ex vivo to 200 ng/ml 8-MOP in combina- sitization in the recipients was observed when PUVA-treated cells tion with 3 J/cm2 UVA light. Untreated and 8-MOP/UVA-treated were obtained from naive donors (Fig. 3C).

cells were injected i.v. into naive syngeneic mice. Twenty-four ϩ hours after injection, a variety of organs, including both the sen- CD11c cells and not T cells are the target for extracorporeal http://classic.jimmunol.org sitized and nonsensitized ears, were excised, weighed, and placed 8-MOP/UVA in gamma-irradiation tubes for counting. Total cpm were deter- We sought to determine which, if any, speciﬁc cell subset was mined, and the percent of cells entering the organs and the cell responsible for the activity of photopheresis. Removal of T cells concentrations were calculated for each organ. The vast majority from the treated cell population by Thy1.2 magnetic bead deple- of cells went to either the liver or the spleen with a minority going tion did not affect the inhibitory capacity of 8-MOP/UVA-treated to lymph nodes and the small intestine (Table I). Approximately cells as long as the same number of cells was used for treatment ϩ

Downloaded from 25% of the counts were not accounted for in the organs we ex- (Fig. 4A). In contrast, depleting CD11c cells resulted in a sig- tracted and are assumed to be distributed in the remaining lym- nificant decrease in the activity of this therapy (Fig. 4B). This phoid and nonlymphoid tissue not unlike the distribution of normal indicates that CD11cϩ cells appear to be the primary and relevant leukocytes. When organ weight was factored in, the concentration cellular target for extracorporeal 8-MOP/UVA in the hapten in the spleen was ϳ10-fold higher. There was no substantial dif- system. ference between 8-MOP/UVA and untreated cells (data not shown), suggesting that the treated cells have similar trafficking as 8-MOP/UVA-induced suppression of CHS can be adoptively normal lymphocytes. We did not detect 8-MOP/UVA-exposed transferred in a hapten-specific fashion cells in the hapten-treated ear (Table I). To determine whether the injection of 8-MOP/UVA-treated cells induces cells with regulatory activity, adoptive transfer experi- Injection of extracorporeally 8-MOP/UVA-treated cells inhibits ments were performed. Mice that received 8-MOP/UVA-treated sensitization of the recipients cells from DNFB-sensitized donors were sensitized with DNFB 5 To establish a model for experimental photopheresis, splenocytes days after injection. Five days after sensitization of the recipients, and lymph node cells of mice previously sensitized against DNFB splenocytes and lymph node cells were obtained and injected i.v. were obtained and exposed ex vivo to 200 ng/ml 8-MOP in com- into naive syngeneic mice (second generation (2° recipients)) (Fig. The Journal of Immunology 5971

Table I. Trafﬁcking of photopheresis-treated cellsa

Weight cpm Percent Injected per Gram Percent Injected

Organ Mean SD Mean SD Mean SD Mean SD

Liver 0.79 0.07 30532 1708 44.95 8.35 35.54 2.91 Spleen 0.09 0.03 30460 17928 398.44 332.95 35.46 30.58 Lymph nodes 0.29 0.08 1611 674 6.49 5.34 1.88 1.15 Kidney 0.28 0.06 1585 86 6.65 2.35 1.85 0.15 Lung 0.16 0.02 332 156 2.43 1.91 0.39 0.27 Large intestines 0.69 0.14 832 551 1.41 1.18 0.97 0.94 Small intestines 1.17 0.18 1089 178 1.08 0.45 1.27 0.30 Stomach 0.20 0.02 202 117 1.15 0.63 0.24 0.20 Blood 0.37 0.06 308 77 0.97 0.37 0.36 0.13 Control ear 0.08 0.01 9 22 0.13 0.38 0.01 0.04 Inﬂamed ear 0.09 0.02 8 19 0.10 0.30 0.01 0.03

a Oxazolone-sensitized BALB/c mice (n ϭ 8) were injected with 2.5 ϫ 106 radiolabeled (average injected dose 85,898 cpm) syngeneic littermate control splenocytes that had been treated with 8-MOP/UVA. On day 1, various organs were harvested, weighed, and counted. Total cpm were measured, and the percent of injected cells entering each organ (percent injected) and the calculated relative cell concentration (percent injected per gram organ weight) were determined.

1). Transfer of cells from 1° recipients that had received cells the activity of the transferred cells. To test this hypothesis, the exposed to 8-MOP/UVA signiﬁcantly suppressed the DNFB re- experiments were repeated in the absence of DNFB sensitization sponse in the 2° recipients (Fig. 5). In contrast, transfer of cells of the 1° recipients. Adoptive transfer studies into 2° recipients from 1° recipients that had received cells exposed to nothing, UVA retained activity upon transfer of cells obtained from the 1° recip-

alone, or 8-MOP alone did not suppress the DNFB response in the ients. However, 2.5 log10 greater cell numbers were required to 2° recipients. The fact that the suppression can be adoptively trans- retain the same suppressive activity (Fig. 8C), suggesting that Ag ferred from 1° recipients into 2° recipients suggests that the infu- is required for optimal activity presumably through increased pro- sion of 8-MOP/UVA-treated cells induces cells with regulatory liferation or activity of the transferred cells. activity in the 1° recipients. To determine whether Ag-specific stimulation is required for in To determine whether these transferred cells cause suppression vivo expansion of Tr, PUVA-treated cells obtained from DNFB- in a hapten-specific fashion, the experiment described in Fig. 5 was sensitized donors were injected into naive recipients. In contrast to repeated. However, in this experiment, the mice were OXA sen- the experiment shown in Fig. 8, recipients were either left un- sitized rather than sensitized with DNFB. Although these animals treated or sensitized against OXA instead of DNFB. Five days retained the ability to suppress DNFB-mediated ear swelling, they after treatment spleen and lymph node cells were obtained and were incapable of decreasing the OXA-mediated ear swelling (Fig. transferred i.v. into naive mice at a concentration of 5 ϫ 107 or 5 by guest on October 1, 2021. Copyright 2005 Pageant Media Ltd. 6). This indicates that the infusion of 8-MOP/UVA-treated spleno- 1 ϫ 10 . 2° recipients were only suppressed upon injection of 5 ϫ cytes obtained from DNFB-sensitized donors induces Ag-specific 107 cells, but not upon injection of 1 ϫ 105 cells (Fig. 9). The same regulatory cells in the 1° recipients. was observed when 1° recipients were left untreated as already shown in Fig. 8C. This implies that expansion of Tr in vivo only 8-MOP/UVA-induced suppression of CHS is mediated by takes place upon Ag-specific boosting. CD4ϩCD25ϩ cells To further characterize the cells involved in transferring the sup- Discussion pression induced by extracorporeal 8-MOP/UVA, we obtained T The data indicate that the infusion of 8-MOP/UVA-treated leuko- cells from 1° recipients that had received 8-MOP/UVA-treated cytes inhibits the immune response in an animal model of CHS via http://classic.jimmunol.org cells obtained from DNFB-sensitized donors. Although i.v. injec- induction of Ag-specific regulation. The most convincing evidence tion of unfractionated cells into naive mice suppressed the sensi- of ECP clinical benefit exists for solid organ transplantation (28) tization of the 2° recipients, no inhibition of the CHS response was and GvHD (29) with more limited reports in a variety of autoim- observed upon injection of either CD4- or CD25-depleted T cells mune and alloimmune disorders (30–32). It is intriguing to spec- (Fig. 7). This indicates that infusion of extracorporeally 8-MOP/ ulate that the mechanistic findings of this study explain these hu- UVA-exposed leukocytes from sensitized mice induces the gener- man results. Ongoing clinical trials are underway to determine

Downloaded from ation of an Ag-specific cell population that expresses CD4 and whether this is indeed the case. We have confirmed the findings of CD25. Ex vivo, these cells produced both IL-10 and TGF-␤ when others that ECP treatment induces the apoptotic cell death of vir- stimulated with hapten in the presence of APC (data not shown). tually all leukocytes (33, 34). Neither 8-MOP nor UVA at this dose To further elucidate the potency of these cells, we sought to were capable of inducing significant cell death. CD3ϩ cells were determine the minimal number of cells required to transfer sup- Ͼ50% annexin Vϩ by day 1, while CD11cϩ cell death was pression. Unfractionated cells obtained from 1° recipients that had slightly delayed. We found that upon reinfusion, these preapop- received extracorporeally 8-MOP/UVA-exposed leukocytes from totic cells are retained primarily in the spleen and liver with sensitized mice were obtained and injected i.v. into naive recipi- smaller amounts retained in the small intestine and secondary ents at various concentrations (Fig. 8, A and B). As few as 1 ϫ 105 lymph nodes. It is presumed that in these sites they begin to ex- total leukocytes induced suppression of DNFB-induced CHS (Fig. press apoptotic-cell-associated molecular patterns and are removed 8B). from the body primarily by resident APC (21). There is growing In the experiments described above, 1° recipients received evidence that clearance or scavenging of apoptotic cells by APC is DNFB topically 5 days after injection of extracorporeally 8-MOP/ not immunologically neutral, as once assumed. In animal models, UVA-treated cells. It was hypothesized that the DNFB treatment it has now been clearly established that clearance of infused apo- of the 1° recipients stimulates the in vivo expansion or increases ptotic cells in the absence of danger signals, irrespective of the cell 5972 PHOTOPHERESIS INDUCES Tr CELLS

FIGURE 4. Extracorporeal 8-MOP/UVA targets CD11cϩ cells but not FIGURE 3. Injection of 8-MOP/UVA treated cells, obtained from T cells. Spleen and lymph node cells were obtained from mice that were DNFB-sensitized mice, inhibits sensitization in the recipients. A and B, sensitized with DNFB. Cells were exposed extracorporeally to 200 ng/ml Spleen and lymph node cells were obtained from mice that were sensitized 8-MOP plus 5 J/cm2 UVA radiation. One group of cells was depleted by with DNFB. Cells were exposed extracorporeally to 200 ng/ml 8-MOP ϩ magnetobead separation of Thy1.2ϩ cells (A), another of CD11cϩ cells (B). 5 J/cm2 UVA radiation. Treated cells were injected i.v. into naive synge- Depleted or bulk cells were injected i.v. into naive syngeneic recipients (1° neic recipients (1° recipients). Five days after injection, recipients were recipients). Five days after injection, recipients were sensitized against sensitized against DNFB and ear challenge performed 5 days later. Positive DNFB and ear challenge performed 5 days later. Positive control mice control mice were sensitized and challenged without injection; negative were sensitized and challenged without injection; negative control mice control mice were ear challenged only. As an additional control, cells were were ear challenged only. Ear swelling was measured 24 h after challenge. Ϫ3 by guest on October 1, 2021. Copyright 2005 Pageant Media Ltd. exposed to 8-MOP alone (A) or UVA alone (B). Ear swelling was measured Ear swelling response is expressed as the difference (centimeters ϫ 10 , 24 h after challenge. Ear swelling response is expressed as the difference mean Ϯ SD) between the thicknesses of the challenged ear compared with Ϫ (centimeters ϫ 10 3, mean Ϯ SD) between the thicknesses of the chal- the thickness of the vehicle-treated ear. lenged ear compared with the thickness of the vehicle-treated ear. C, Spleen and lymph node cells were obtained either from naive mice (No sensi) or from mice that were sensitized with DNFB (Sensi). Cells were exposed extracorporeally to 200 ng/ml 8-MOP plus 5 J/cm2 UVA light. regulatory activity. This activity was lost when non-APC cells Cells were injected identically as in A and B. alone were used (A. Krutsick, J. Huber, K. Campbell, and D. Peritt, unpublished observations). It appears that the action of 8-MOP/UVA-treated cells may be multifunctional. In certain cir- http://classic.jimmunol.org type (25, 35–40) or the means of apoptotic induction (23, 24, cumstances such as this haptenated model it is essential that APCs 41–43), induces immune tolerance (44, 45). It has been speculated carrying the Ag directly stimulate regulatory cells. In other situa- that this immune modulation evolved as a system to constantly tions, when the Ag is readily available in the host, the delivery of sample self Ag in the periphery and to inhibit immune autoreac- apoptotic cells may be disconnected from the Ag because they will tivity (26). Thus, infusion of apoptotic cells may simply be a meet in the secondary lymph node system. This latter system method to harness a natural process for therapeutic treatment of seems more likely the mechanism in diseases such as solid organ

Downloaded from disease. The current view is that the apoptotic-cell-associated mo- transplantation. What is clear is that the action of apoptotic cells is lecular pattern receptors on the surface of APCs recognize and not direct at the site of inflammation because ECP-treated cells engage apoptotic cells (21). This engagement leads to a series of could not be detected in the inflamed ears. events including phagocytic uptake of the apoptotic cells and sig- For cutaneous UVB-induced immunomodulation there is good naling by a subset of these receptors. This signaling has been as- evidence that UVB-damaged cutaneous APC are involved in this sociated with a decreased ability to efficiently stimulate T effector process. When Langerhans cells are exposed to UVB they migrate cell responses (46), increased anti-inflammatory cytokine produc- into the draining lymph nodes and are not able to present the hap- tion (47, 48), decreased proinflammatory cytokine production (23, ten properly, thereby failing to appropriately sensitize (49). There 35), and possibly aid the generation of Tr (11, 20). is evidence that this treatment directly stimulates regulatory cell Our data suggest that ECP-treated CD11cϩ cells, which carry generation, which is transferable to naive animals and suppresses the hapten Ag, may have the ability to directly tolerize before their future immune responses in an Ag-specific fashion (50). Reduction eventual demise. Using nonhaptenated CD11cϩ cells or cells from of DNA damage by exogenous repair enzymes restores the immu- animals sensitized 3 wk earlier did not induce immune tolerance nostimulatory capacity of APC obtained from lymph nodes drain- (data not shown). In vitro we were able to demonstrate that human ing UVB-exposed skin and the development of Tr is blocked upon APCs treated with ECP induced the generation of CD4 cells with administration of the DNA repair enzymes (51). IL-12, which is The Journal of Immunology 5973

FIGURE 5. Injection of 8-MOP/UVA-treated cells obtained from DNFB-sensitized mice induces transferable tolerance in recipients. Spleen FIGURE 6. Regulatory cells induced by extracorporeal 8-MOP/UVA and lymph node cells were obtained from mice that were sensitized with suppress in an Ag-speciﬁc fashion. Spleen and lymph node cells were DNFB. Cells were exposed extracorporeally to 200 ng/ml 8-MOP plus 5 obtained from mice that were sensitized with DNFB. Cells were exposed 2 J/cm2 UVA radiation. Treated cells were injected i.v. into naive syngeneic extracorporeally to 200 ng/ml 8-MOP plus 5 J/cm UVA radiation. Treated recipients (1° recipients). As controls, cells were exposed only to 8-MOP cells were injected i.v. into naive syngeneic recipients (1° recipients). Five without UVA (8-MOP). Treated cells were injected i.v. into naive synge- days after injection, recipients were sensitized against DNFB, and ear chal- neic mice. Five days after injection, recipients were sensitized against lenge was performed 5 days later. Spleen and lymph node cells were ob- DNFB. Five days thereafter, spleen and lymph node cells were obtained tained from the recipient mice and transferred i.v. into a second generation from the recipient mice and transferred i.v. into a second generation of of syngeneic naive mice (2° recipients). Twenty-four hours after transfer, syngeneic naive mice (2° recipients). Mice were sensitized against DNFB mice were sensitized against DNFB or OXA. Ear challenge with DNFB 24 h after transfer, and ear challenge performed 5 days thereafter. Positive and OXA was performed 5 days later. Positive control mice were sensitized control mice were sensitized and challenged without transfer; negative con- and challenged without transfer; negative control mice were ear challenged trol mice were ear challenged only. Ear swelling was measured 24 h after only. Ear swelling was measured 24 h after challenge. Ear swelling re- Ϫ3 challenge. Ear swelling response is expressed as the difference (centime- sponse is expressed as the difference (centimeters ϫ 10 , mean Ϯ SD) ters ϫ 10Ϫ3, mean Ϯ SD) between the thicknesses of the challenged ear between the thicknesses of the challenged ear compared with the thickness compared with the thickness of the vehicle-treated ear. of the vehicle-treated ear.

able to prevent UVB-induced immunosuppression (52), may work in part via its capacity to reduce UVB-mediated DNA damage (53, ilar to the UVB-damaged Langerhans cells of the skin. Both the 54). Taken together, all these ﬁndings indicate that both the inhi- cutaneous UVB and the 8-MOP/UVA treated APC carry the hap- bition of the induction of CHS and the generation of Tr are a ten to the lymphoid organs. This hypothesis is supported by the consequence of UVB-induced DNA damage in APC. Because, in observation that we do not induce suppression in the recipients contrast with the cutaneous UVB treatment, the 8-MOP/UVA when we inject PUVA-treated cells obtained from donors which

by guest on October 1, 2021. Copyright 2005 Pageant Media Ltd. treatment is ex vivo, it allows for an easier dissection of the system had been sensitized 3 wk earlier (data not shown). to determine the mechanism of action. It is possible that in a nonhaptenated system, any apoptotic cell Although the functional characterization of clinical response to will induce tolerance, but in the case of haptens, the Ag must be ECP is quite advanced and there is information on the downstream linked on the apoptotic cell and only APC retain this ability fol- immune changes, the initiating events are not understood at all. In lowing 5 days in vivo. This latter mechanism seems likely because particular, we were interested which, if any, cell type might be apoptotic cells from pure T cells, cell lines, or even xenogeneic targeted by 8-MOP/UVA. In this system we only observed immu- neutrophils have been shown to induce immune tolerance in other nomodulation by injecting 8-MOP/UVA-treated cells obtained nonhapten-based systems. This suggests that, in contrast to the from DNFB-sensitized donors, which led to the Ag specificity. We hapten system, treated APCs may not be critical for the induction http://classic.jimmunol.org were quite surprised to see this activity even in cells obtained 5 of tolerance and a disconnect between the apoptotic cell and the days after sensitization. It was surmised that DNFB-specific T ef- tolerizing Ag is possible. fector cells might be the primary target for 8-MOP/UVA, and that In human clinical trials immunophenotypic analysis of dendritic infusion of apoptotic DNFB-specific T effector cells might cause cell (DC) populations revealed a preponderance of DC1 monocytic the induction of an anti-clonotypic T cell response, as has been DCs in all patients before the initiation of ECP (55). Nine of 10 suggested as a mechanism (7). However, when we depleted the patients demonstrated a shift from DC1 to DC2 and a concordant ϩ Downloaded from spleen and lymph node cell suspension of Thy1.2 cells before shift from a predominantly Th1 to Th2 cytokine profile after ECP, 8-MOP/UVA exposure, we still observed an inhibition in the im- which would suggest that ECP alters alloreactivity by affecting mune response. When cells were depleted of CD11cϩ cells, sup- allotargeted effector T cells and Ag-presenting DC. This study has pression was lost and suppression could not be transferred into the been criticized because the definition for DC used was in contra- secondary recipient when treated splenocytes were depleted of diction to the standard classification of DC, the expansion was CD11cϩ cells, indicating that Tr had not developed in the 1° re- conducted ex vivo, and certain controls were missing (56). Nev- cipients (data not shown). This data implies that while all cells may ertheless, this study and others favor the concept of the develop- undergo apoptosis following extracorporeal 8-MOP/UVA therapy, ment of active specific rather than suppressive nonspecific immune APCs are critically important in this system. In addition, suppres- responses induced by ECP (5, 57) the exact nature of which needs sion was only observed upon PUVA exposure of cells obtained to be determined. from sensitized but not from naive mice. We propose that extra- One of the primary mechanisms by which these tolerogenic corporeal 8-MOP/UVA treatment of APC still carrying the hapten APC may operate is via cytokine modulation. There is a large induces an apoptotic cascade, which may take place over several volume of literature from in vitro and some in vivo work showing days. Infusion of these damaged, slowly dying APC does not in- apoptotic cell induction of anti-inflammatory cytokines and inhi- duce sensitization but in contrast causes immunomodulation sim- bition of proinflammatory cytokines by APC (reviewed in Ref. 5974 PHOTOPHERESIS INDUCES Tr CELLS

FIGURE 7. 8-MOP/UVA-induced regulatory T cells express CD4 and CD25. Spleen and lymph node cells were obtained from mice that were sensitized with DNFB. Cells were exposed extracorporeally to 200 ng/ml 8-MOP plus 5 J/cm2 UVA radiation. Treated cells were injected i.v. into naive syngeneic recipients (1° recipients), which were sensitized against DNFB 5 days later. Five days thereafter, bulk splenocytes and lymph node cells were obtained from the recipient mice and transferred i.v. into a second generation of syngeneic naive mice (2° recipients). Cells were depleted ϩ ϩ of either CD4 cells (A) or CD25 cells (B) by magnetobead separation. FIGURE 8. Regulatory cells induced by 8-MOP/UVA suppress in a Mice were sensitized against DNFB 24 h after transfer, and ear challenge

by guest on October 1, 2021. Copyright 2005 Pageant Media Ltd. dose-dependent manner. Spleen and lymph node cells were obtained from was performed 5 days thereafter. Positive control mice were sensitized and mice that were sensitized against DNFB. Cells were exposed extracorpo- challenged without transfer; negative control mice were ear challenged really to 8-MOP plus UVA (PUVA). PUVA-treated cells were injected i.v. only. Ear swelling was measured 24 h after challenge. Ear swelling re- into naive syngeneic mice. Five days after injection, recipients were sen- ϫ Ϫ3 Ϯ sponse is expressed as the difference (centimeters 10 , mean SD) sitized against DNFB. Five days thereafter, lymph node cells were obtained between the thicknesses of the challenged ear compared with the thickness from the recipient mice and transferred i.v. at concentrations ranging from of the vehicle-treated ear. 5 ϫ 107 to 1 ϫ 105 (A) and from 1 ϫ 105 to 1 ϫ 104 (B) into a second generation of syngeneic naive mice (2° recipients). In addition, cells were obtained from 1° recipients, which received PUVA-treated cells but were 21). This increase in anti-inflammatory and decrease in proinflam- not sensitized with DNFB (C). Ten days after injection, lymph node cells http://classic.jimmunol.org matory cytokines may, in part, be responsible for the clinical ef- were obtained and transferred into 2° recipients at concentrations ranging fects of ECP seen in humans and animals. The relative importance from 5 ϫ 107 to 1 ϫ 105. Twenty-four hours after transfer, mice were of these cytokine alterations vs the generation of regulatory cell sensitized against DNFB. Ear challenge with DNFB was performed 5 days activity that we describe in this study is still not clear. thereafter. Positive control mice were sensitized and challenged without transfer; negative control mice were ear challenged only. Ear swelling was In humans, evidence also suggests that ECP modulates cytokine measured 24 h after challenge. Ear swelling response is expressed as the secretion. There has been a reported decrease in proinflammatory difference (centimeters ϫ 10Ϫ3, mean Ϯ SD) between the thicknesses of

Downloaded from ␣ ␥ cytokines TNF- and IFN- in CTCL and GvHD patients under- the challenged ears compared with the thickness of the vehicle-treated ears. going ECP therapy (58). This decrease occurs in cells other than the apoptotic (treated cells), suggesting a systemic effect on recipient cells not directly treated by ECP (59). Craciun et al. (57) have reported an increase in the anti-inflammatory cytokines IL-10 and It was shown Ͼ25 years ago that the topical application of con- IL-1 receptor antagonist. Gorgun et al. (55), using an ex vivo cul- tact allergens (haptens) does not induce sensitization when the ture system, found an increase in IL-4 and IL-10. In vitro produc- hapten is applied onto UVB-exposed skin (62). In addition, indi- tion of immunosuppressive cytokines, including IL-10 following viduals having received the allergen on UVB-exposed skin cannot ingestion of apoptotic cells, has been observed (48) and may be be sensitized later against the very same hapten, implying that involved in the earlier therapeutic benefit reported in ECP (60). hapten-specific tolerance had developed (50). This tolerance is me- Others have also suggested systemic effects on leukocyte popula- diated, in part, via the induction of Ag-specific Tr, because sup- tions and function (55, 61). These data correspond to our ex vivo pression can be adoptively transferred into naive recipients (63). IL-10 production data reported in this study and our recent in vivo According to recent observations, UV-induced Tr belong to the description showing a role for IL-10 in an UVB-induced Tr CD4ϩ/CD25ϩ subtype (18), express CTLA-4 (64), bind the lectin system (18). dectin-2 (63), and release IL-10 upon antigenic stimulation (64). The Journal of Immunology 5975

induces immunologic tolerance. This tolerance decreases immune response to subsequent hapten challenge. The nature of this tolerance is due primarily to Tr because transfer conferred similar protection. These regulatory cells are CD4 and CD25 positive and ex vivo produce IL-10 and TGF-␤. The effector mechanism by which these cells inhibit hapten responses is an area of current investigation. The demonstration of the induction of Tr may explain why, in humans, ECP exerts a beneficial effect in a wide variety of diseases which would be amenable to such activity. The generation of Ag-specific Tr may explain why generalized immunosuppression has not been noted with ECP. Further investigation into the FIGURE 9. In vivo expansion of Tr cells induced by 8-MOP/UVA is mechanism by which photopheresis may induce immunological hapten-specific. Spleen and lymph node cells were obtained from mice that tolerance will determine ways to better use the current therapy and were sensitized against DNFB. Cells were exposed extracorporeally to indicate ways in which improvements can be made for the benefit 8-MOP plus UVA (PUVA). PUVA-treated cells were injected i.v. into of our patients. naive syngeneic mice. Five days after injection, recipients were sensitized against OXA. Five days thereafter, lymph node cells were obtained from Acknowledgments the recipient mice and transferred i.v. into naive 2° recipients. In addition, We are grateful to Eva Emmell and Terry Goletz for conducting the ra- cells were obtained from 1° recipients, which received PUVA-treated cells diolabelled cell tracking study and Stefan Beissert for critically reading the ϫ 7 ϫ but were not sensitized with DNFB (naive), and injected (5 10 or 1 manuscript. 105 cells) into naive 2° recipients. Twenty-four hours after transfer, mice were sensitized against DNFB. Ear challenge with DNFB was performed Disclosures 5 days thereafter. Positive control mice were sensitized and challenged D. Peritt is an employee of, and T. Schwarz received grant support from, without transfer; negative control mice were ear challenged only. Ear Therakos, a Johnson & Johnson company. swelling was measured 24 h after challenge. Ear swelling response is ex- Ϫ3 pressed as the difference (centimeters ϫ 10 , mean Ϯ SD) between the References thicknesses of the challenged ears compared with the thickness of the ve- 1. Gilchrest, B. A., J. A. Parrish, L. Tanenbaum, H. A. Haynes, and hicle-treated ears. T. B. Fitzpatrick. 1976. Oral methoxsalen photochemotherapy of mycosis fun- goides. Cancer 38: 683–689. 2. Efferth, T., U. Fabry, and R. Osieka. 2001. Induction of apoptosis, depletion of Unfortunately, the complexity of the cutaneous system has con- glutathione, and DNA damage by extracorporeal photochemotherapy and psor- founded the mechanistic interpretation, and we are still left with an alen with exposure to UV light in vitro. Anticancer Res. 21: 2777–2783. 3. Edelson, R., C. Berger, F. Gasparro, B. Jegasothy, P. Heald, B. Wintroub, incomplete explanation of this phenomenon (65). E. Vonderheid, R. Knobler, K. Wolff, G. Plewig, et al. 1987. Treatment of cu- To get an idea whether ECP might also induce Tr, we set up an taneous T-cell lymphoma by extracorporeal photochemotherapy: preliminary re- experimental in vivo model for ECP using CHS as a readout sys- sults. N. Engl. J. Med. 316: 297–303. 4. Heald, P., A. Rook, M. Perez, B. Wintroub, R. Knobler, B. Jegasothy, tem. With this model we demonstrate that extracorporeal exposure F. Gasparro, C. Berger, and R. Edelson. 1992. Treatment of erythrodermic cu-

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