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The Journal of Immunology, 2005, 174: 5968-5976.
Copyright © 2005 by The American Association of Immunologists

Intravenous Infusion of Syngeneic Apoptotic Cells by Photopheresis Induces Antigen-Specific Regulatory T Cells 1

Akira Maeda*, Agatha Schwarz*, Kerstin Kernebeck*, Nicole Gross*, Yoshinori Aragane{dagger}, David Peritt{ddagger} and Thomas Schwarz2,§

* Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Department of Dermatology, University Münster, Münster, Germany; {dagger} Department of Dermatology, Kinki University School of Medicine, Osakasayama-City, Osaka, Japan; {ddagger} Therakos, Exton, PA 19341; and § Department of Dermatology, University Kiel, Kiel, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 remains 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 immunomodulatory effects via the induction of Ag-specific regulatory T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The introduction of photochemotherapy (psoralen plus UVA (PUVA) 3), which consists of the oral ingestion of the photosensitizer 8-methoxypsoralen (8-MOP) followed by total body UVA (320–400 nm) exposure, has had tremendous impact for the therapy of cutaneous T cell lymphoma (CTCL) (1) and is still widely used. The mechanism of PUVA is thought to be primarily depletion of malignant T cells from the skin (2). Early studies in CTCL patients considered the idea of a pheresis procedure bringing the malignant cells outside the body for PUVA treatment. This formed the basis for the development of extracorporeal photopheresis (ECP). Surprisingly, several of the first patients treated with photopheresis went into partial or complete remission (3, 4). However, the beneficial effect could not be explained by a simple destruction of malignant cells because only a small proportion of pathogenic T cells are treated during an extracorporeal cycle. This led to the supposition that an immune-mediated systemic anti-tumor-specific activity may be involved (5). Current ECP treatments involve a closed-loop, sterile, patient-connected, point of care device withdrawing ~5 billion peripheral blood leukocytes from the patient by apheresis followed by incubation of the cells with 8-MOP and UVA exposure in an extracorporeal setting.

Because of its safety and efficacy in the treatment of CTCL, which is based on experience with this regimen for nearly 20 years, ECP has been tried in a variety of diseases that have a suspected involvement of pathogenic T cells, including rejection of organ transplants, graft-vs-host disease (GvHD) and autoimmune disorders (6). The efficacy and mechanism of action in CTCL has been suggested to be based upon an anti-idiotypic TCR vaccination (7). Dying tumor cells are ingested by APC and induce an Ag (monoclonal TCR)-specific CTL-mediated immune response against the tumor. This hypothesis has recently led to a concept to improve the efficacy of photopheresis by incubating treated cells overnight to lengthen the time apoptotic CTCL cells interact with treated APC before reinfusion. This intriguing hypothesis, termed transimmunization (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 these diseases are extremely heterogeneous clinically, they share similar underlying pathophysiological mechanisms and response to immunosuppressive therapies. It is unlikely that ECP induces a generalized immunosuppression because patients, including those undergoing long-term ECP therapy, have no reported higher risk of developing infections or malignancies (9) and respond normally to both novel and recall Ag (10). More recently, it has been suggested that ECP may induce Ag-specific immunomodulation, possibly via regulatory T cells (Tr) (11).

Tr comprise a heterogeneous group of T lymphocytes, which actively inhibit immune responses (12, 13, 14). They have been recognized to play an important role in the prevention of autoimmunity, GvHD, and transplant rejection (15, 16, 17). Clinically, there is great excitement about the potential to develop methodologies that can harness Tr for therapeutic activity. The complexity introduced by the varying types and activities of Tr has added an additional challenge. Solar/UV radiation, in particular the midwave range (UVB, 290–320 nm), long has been recognized to exhibit the capacity to induce immunotolerance, in part, via Ag-specific Tr (18, 19).

Over the last few years it has been well documented that apoptotic cell clearance by APC may regulate immune responses via modulation of APC (20, 21, 22). Although the majority of this work has been conducted in vitro, excellent efficacy and mechanistic studies have recently been reported in murine models of heart allograft rejection (23), response to haptens (24), OVA, and LPS-induced lung inflammation (25). The cellular and molecular mechanisms of this tolerance induction have been partially identified. A subset of apoptotic receptors, responsible for recognition and phagocytosis of apoptotic cells, also signal the APC to decrease proinflammatory cytokines, increase anti-inflammatory cytokines and functionally inhibit the ability to stimulate effector T cells (21). Recent suggestions have been made that Tr may be generated following APC engagement of apoptotic cells (22). It is now clear that ingestion of apoptotic cells is not a neutral event as once thought, but an active process of immune tolerance induction, the exact nature of which is unfolding quickly. Teleologically, this process has been described as another peripheral immune tolerance mechanism. Autologous cells, naturally undergoing apoptosis in the periphery, are ingested by resident phagocytes and migrate to draining lymphatics. Here they act as a constant reminder to roving T cells about self (26). Only in the presence of danger signals will the phagocyte be altered to become immunogenic and begin the appropriate immune cascade to fight pathogens (27).

In the context of these studies, we were interested in investigating whether ECP exhibits the capacity to induce Ag-specific Tr. For this purpose, we established an in vivo model for experimental photopheresis. Using this model, we demonstrated unresponsiveness in treated mice that was transferable to naive mice, suggesting the involvement of a regulatory cell population. This transferable protection is Ag specific, dose dependent, and requires CD4+ and CD25+ cells. Taken together, these data provide evidence that photopheresis may exhibit the capacity to induce Ag-specific regulatory cells. These findings may further add to the understanding of the mode of action of ECP, provide an explanation for the long-lived efficacy and safety of ECP in the treatment of a variety of diseases, and open up the possibility of better clinical use and future improvements in this therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and reagents

C3H/HeN mice (8–10 wk of age) were purchased from Harlan Winkelmann. BALB/c female mice (4–6 wk of age) were purchased from Charles River Laboratories. Animals were housed under specific pathogen-free conditions, and animal care was provided by expert personnel in compliance with the relevant laws and institutional guidelines. 2,4-Dinitrofluorobenzene (DNFB) and oxazolone (OXA) were purchased from Sigma-Aldrich. UVADEX (8-MOP) was provided from Therakos, a Johnson & Johnson company (Exton, PA).

Trafficking of photopheresis-treated cells

BALB/c mice (n = 6) were sensitized on day –5 by topical application of 20 µl of a 1.6% (w/v) solution of OXA in ethanol on both the inside and outside of the left ear. On day 0, animals were challenged with 20 µl of 0.8% solution of OXA on the inside and outside of the left ear while the right ear was treated with vehicle.

On day 0, whole splenocytes from syngeneic littermate mice were pooled, resuspended in medium, and radiolabeled using standard laboratory procedures. Cells were labeled with 51Cr (100 µCi 51Cr per ~2 x 107 cells) for 1 h at 37°C, washed twice with HBSS, resuspended at 12.5 x 106 cells/ml, and placed in a T-75 flask. To this flask, 200 ng/ml 8-MOP was added before UVA irradiation (3 J/cm2). Cells were quickly removed from the flask to avoid adherence and placed at the appropriate concentration for injection. Mice were treated with ECP-treated cells via tail-vein injection with 0.2 ml (~2.5 x 106 cells) of 51Cr labeled cells. The mice were then euthanized via CO2 asphyxiation at 24 h. Various organs (lungs, spleen, thymus, liver, stomach, lymph nodes, kidneys, small and large intestines, sensitized and unsensitized ears) were harvested, placed in individual counting tubes, weighed, and counted for radioactivity using an LKB gamma counter. Total cpm were measured, and the percent of injected cells entering the organs and cell concentration were calculated.

Experimental photopheresis

Donor mice were sensitized against DNFB. Twenty-four hours later, spleens and regional lymph nodes were removed and single-cell suspensions 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 UVA irradiation, a UVA high-power device (Sellamed 4000; Sellas Sunlight) was used with an emission peak at 365 nm (output 40 mW/cm2). After washing in PBS, the cell number was adjusted to 2.5 x 108/ml and 200 µl of cells were injected i.v. into naive syngeneic mice. Five days later, recipients were sensitized by application of 50 µl of 0.5% DNFB. Recipients were challenged by application of 0.3% DNFB on the left ear 5 days after sensitization. After another 24 h, the ear swelling response was measured.

Cell viability

Normal donor mice were used to obtain cells from spleens and regional lymph nodes, and single cell suspensions were prepared. After washing, cells were incubated with 200 ng/ml 8-MOP for 30 min in the dark, followed by exposure to UVA radiation (5 J/cm2). These cells were incubated under standard conditions at 2 x 106 cells/ml in RPMI supplemented with 10% FCS. Every day for 5 days an aliquot was removed and stained with annexin V FITC and both CD3 PerCP and CD11c allophycocyanin. These samples were analyzed by flow cytometry (EPICS; Coulter). Cell death is portrayed as a line graph of annexin V-positive cells in either the CD3+ or CD11c+ populations.

Contact hypersensitivity (CHS)

C3H/HeN mice were sensitized by painting 50 µl of DNFB (0.5% in acetone/olive oil, 4:1) on the shaved back on day 0. On day 5, 20 µl of 0.3% DNFB was applied on the left ear, the vehicle acetone/olive oil was used on the right ear as a control. OXA was applied at a concentration of 2% for sensitization and at 0.5% for elicitation. Ear swelling was measured in a blinded fashion with a spring-loaded micrometer (Mitutoyo, Kawasaki, 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-treated ear in sensitized animals and was expressed in centimeters x 10–3 (mean ± SD). Mice that were ear-challenged without prior sensitization served as negative controls. Each group consisted of at least seven mice. Each experiment was performed at least twice.

Adoptive cell transfer

Spleens and regional lymph nodes were removed from mice that had received 8-MOP/UVA-treated splenocytes (1° recipients). The cell number was adjusted to 2.5 x 108/ml and cells were injected i.v. (200 µl) into naive syngeneic mice (2° recipients). Twenty-four hours later, 2° recipients were sensitized against DNFB. Five days later, ear challenge was performed and ear swelling was measured 24 h thereafter. The scheme of experimental photopheresis, including the transfer studies, is demonstrated in Fig. 1.



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  		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.

 
Depletion of subpopulations

For depletion of subpopulations, lymphocytes obtained from regional lymph nodes and spleens were incubated with microbeads coated with Ab against CD4, CD8, CD25, CD11c, or Thy1.2 (Miltenyi Biotec). The incubation was followed by negative selection via magnetic bead separation using the autoMACS system (Miltenyi Biotec). The negative fractions were harvested, washed, and adjusted to the appropriate cell numbers and subsequently used for injection. The efficacy of depletion or isolation was determined by flow cytometric analysis (EPICS; Coulter).

Statistical analysis

All statistics were analyzed by single tail Student’s t test. **, p < 0.005; *, p < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
8-MOP/UVA induces apoptosis

Splenocytes from control mice were incubated with 8-MOP and exposed to UVA radiation and incubated under standard conditions for up to 5 days. These cells were analyzed by flow cytometry 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 CD3+ and CD11c+ subsets. Interestingly, the CD11c+ cells had a slightly slower kinetic of annexin V positivity as compared with CD3+ cells (Fig. 2). We have also noted a similar delay in human cell death following ECP (data not shown).



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  		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 UVA radiation (5 J/cm2). Every day for 5 days, an aliquot was removed and stained with annexin V FITC and both CD3 PerCP and CD11c APC. A, Linear graph of the percent total (squares), CD3 (diamonds), and CD11c (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 5 forward/side scatter and CD11c/annexin V graphs.

 
Extracorporeally 8-MOP/UVA-treated cells are retained primarily in the spleen and liver

Splenocytes from syngeneic littermate control mice were labeled with 51Cr and exposed ex vivo to 200 ng/ml 8-MOP in combination with 3 J/cm2 UVA light. Untreated and 8-MOP/UVA-treated cells were injected i.v. into naive syngeneic mice. Twenty-four hours after injection, a variety of organs, including both the sensitized and nonsensitized ears, were excised, weighed, and placed in gamma-irradiation tubes for counting. Total cpm were determined, and the percent of cells entering the organs and the cell concentrations were calculated for each organ. The vast majority of cells went to either the liver or the spleen with a minority going to lymph nodes and the small intestine (Table I). Approximately 25% of the counts were not accounted for in the organs we extracted and are assumed to be distributed in the remaining lymphoid and nonlymphoid tissue not unlike the distribution of normal leukocytes. When organ weight was factored in, the concentration in the spleen was ~10-fold higher. There was no substantial difference between 8-MOP/UVA and untreated cells (data not shown), suggesting that the treated cells have similar trafficking as normal lymphocytes. We did not detect 8-MOP/UVA-exposed cells in the hapten-treated ear (Table I).


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  		Table I. Trafficking of photopheresis-treated cellsa

 
Injection of extracorporeally 8-MOP/UVA-treated cells inhibits sensitization of the recipients

To establish a model for experimental photopheresis, splenocytes and lymph node cells of mice previously sensitized against DNFB were obtained and exposed ex vivo to 200 ng/ml 8-MOP in combination with 5 J/cm2 UVA light and injected i.v. into naive syngeneic mice. These recipients were sensitized with DNFB, and ear challenge was performed 5 days later. Ear swelling was measured 24 h after challenge (Fig. 1). Recipients of 8-MOP/UVA-treated cells had a significantly decreased ear swelling response compared with controls (Fig. 3). In contrast, when cells were exposed to either 8-MOP (Fig. 3A) or UVA alone (Fig. 3B), ear swelling of the recipients was not affected. In addition, no suppression of sensitization in the recipients was observed when PUVA-treated cells were obtained from naive donors (Fig. 3C).



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  		FIGURE 3. Injection of 8-MOP/UVA treated cells, obtained from DNFB-sensitized mice, inhibits sensitization in the recipients. A and B, Spleen and lymph node cells were obtained from mice that were sensitized with DNFB. Cells were exposed extracorporeally to 200 ng/ml 8-MOP + 5 J/cm2 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. Positive control mice were sensitized and challenged without injection; negative control mice were ear challenged only. As an additional control, cells were exposed to 8-MOP alone (A) or UVA alone (B). Ear swelling was measured 24 h after challenge. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thicknesses of the challenged 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. Cells were injected identically as in A and B.

 
CD11c+ cells and not T cells are the target for extracorporeal 8-MOP/UVA

We sought to determine which, if any, specific cell subset was responsible for the activity of photopheresis. Removal of T cells from the treated cell population by Thy1.2 magnetic bead depletion did not affect the inhibitory capacity of 8-MOP/UVA-treated cells as long as the same number of cells was used for treatment (Fig. 4A). In contrast, depleting CD11c+ cells resulted in a significant decrease in the activity of this therapy (Fig. 4B). This indicates that CD11c+ cells appear to be the primary and relevant cellular target for extracorporeal 8-MOP/UVA in the hapten system.



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  		FIGURE 4. Extracorporeal 8-MOP/UVA targets CD11c+ cells but not T cells. 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. One group of cells was depleted by magnetobead separation of Thy1.2+ cells (A), another of CD11c+ cells (B). Depleted or bulk 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. Positive control mice were sensitized and challenged without injection; negative control mice were ear challenged only. Ear swelling was measured 24 h after challenge. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thicknesses of the challenged ear compared with the thickness of the vehicle-treated ear.

 
8-MOP/UVA-induced suppression of CHS can be adoptively transferred in a hapten-specific fashion

To determine whether the injection of 8-MOP/UVA-treated cells induces cells with regulatory activity, adoptive transfer experiments were performed. Mice that received 8-MOP/UVA-treated cells from DNFB-sensitized donors were sensitized with DNFB 5 days after injection. Five days after sensitization of the recipients, splenocytes and lymph node cells were obtained and injected i.v. into naive syngeneic mice (second generation (2° recipients)) (Fig. 1). Transfer of cells from 1° recipients that had received cells exposed to 8-MOP/UVA significantly suppressed the DNFB response in the 2° recipients (Fig. 5). In contrast, transfer of cells from 1° recipients that had received cells exposed to nothing, UVA alone, or 8-MOP alone did not suppress the DNFB response in the 2° recipients. The fact that the suppression can be adoptively transferred from 1° recipients into 2° recipients suggests that the infusion of 8-MOP/UVA-treated cells induces cells with regulatory activity in the 1° recipients.



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  		FIGURE 5. Injection of 8-MOP/UVA-treated cells obtained from DNFB-sensitized mice induces transferable tolerance in recipients. 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). As controls, cells were exposed only to 8-MOP without UVA (8-MOP). Treated cells were injected i.v. into naive syngeneic mice. Five days after injection, recipients were sensitized against DNFB. Five days thereafter, 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). Mice were sensitized against DNFB 24 h after transfer, and ear challenge performed 5 days thereafter. Positive control mice were sensitized and challenged without transfer; negative control mice were ear challenged only. Ear swelling was measured 24 h after challenge. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thicknesses of the challenged ear compared with the thickness of the vehicle-treated ear.

 
To determine whether these transferred cells cause suppression in a hapten-specific fashion, the experiment described in Fig. 5 was repeated. However, in this experiment, the mice were OXA sensitized rather than sensitized with DNFB. Although these animals retained the ability to suppress DNFB-mediated ear swelling, they were incapable of decreasing the OXA-mediated ear swelling (Fig. 6). This indicates that the infusion of 8-MOP/UVA-treated splenocytes obtained from DNFB-sensitized donors induces Ag-specific regulatory cells in the 1° recipients.



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  		FIGURE 6. Regulatory cells induced by extracorporeal 8-MOP/UVA suppress in an Ag-specific fashion. 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). Five days after injection, recipients were sensitized against DNFB, and ear challenge was performed 5 days later. 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 or OXA. Ear challenge with DNFB and OXA was performed 5 days later. Positive control mice were sensitized and challenged without transfer; negative control mice were ear challenged only. Ear swelling was measured 24 h after challenge. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thicknesses of the challenged ear compared with the thickness of the vehicle-treated ear.

 
8-MOP/UVA-induced suppression of CHS is mediated by CD4+CD25+ cells

To further characterize the cells involved in transferring the suppression induced by extracorporeal 8-MOP/UVA, we obtained T cells from 1° recipients that had received 8-MOP/UVA-treated cells obtained from DNFB-sensitized donors. Although i.v. injection of unfractionated cells into naive mice suppressed the sensitization of the 2° recipients, no inhibition of the CHS response was observed upon injection of either CD4- or CD25-depleted T cells (Fig. 7). This indicates that infusion of extracorporeally 8-MOP/UVA-exposed leukocytes from sensitized mice induces the generation of an Ag-specific cell population that expresses CD4 and CD25. Ex vivo, these cells produced both IL-10 and TGF-{beta} when stimulated with hapten in the presence of APC (data not shown).



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  		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 of either CD4+ cells (A) or CD25+ cells (B) by magnetobead separation. Mice were sensitized against DNFB 24 h after transfer, and ear challenge was performed 5 days thereafter. Positive control mice were sensitized and challenged without transfer; negative control mice were ear challenged only. Ear swelling was measured 24 h after challenge. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thicknesses of the challenged ear compared with the thickness of the vehicle-treated ear.

 
To further elucidate the potency of these cells, we sought to determine the minimal number of cells required to transfer suppression. Unfractionated cells obtained from 1° recipients that had received extracorporeally 8-MOP/UVA-exposed leukocytes from sensitized mice were obtained and injected i.v. into naive recipients at various concentrations (Fig. 8, A and B). As few as 1 x 105 total leukocytes induced suppression of DNFB-induced CHS (Fig. 8B).



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  		FIGURE 8. Regulatory cells induced by 8-MOP/UVA suppress in a dose-dependent manner. Spleen and lymph node cells were obtained from mice that were sensitized against DNFB. Cells were exposed extracorporeally to 8-MOP plus UVA (PUVA). PUVA-treated cells were injected i.v. into naive syngeneic mice. Five days after injection, recipients were sensitized against DNFB. Five days thereafter, lymph node cells were obtained from the recipient mice and transferred i.v. at concentrations ranging from 5 x 107 to 1 x 105 (A) and from 1 x 105 to 1 x 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 not sensitized with DNFB (C). Ten days after injection, lymph node cells were obtained and transferred into 2° recipients at concentrations ranging from 5 x 107 to 1 x 105. Twenty-four hours after transfer, mice were sensitized against DNFB. Ear challenge with DNFB was performed 5 days thereafter. Positive control mice were sensitized and challenged without transfer; negative control mice were ear challenged only. Ear swelling was measured 24 h after challenge. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thicknesses of the challenged ears compared with the thickness of the vehicle-treated ears.

 
In the experiments described above, 1° recipients received DNFB topically 5 days after injection of extracorporeally 8-MOP/UVA-treated cells. It was hypothesized that the DNFB treatment of the 1° recipients stimulates the in vivo expansion or increases the activity of the transferred cells. To test this hypothesis, the experiments were repeated in the absence of DNFB sensitization of the 1° recipients. Adoptive transfer studies into 2° recipients retained activity upon transfer of cells obtained from the 1° recipients. However, 2.5 log10 greater cell numbers were required to retain the same suppressive activity (Fig. 8C), suggesting that Ag is required for optimal activity presumably through increased proliferation or activity of the transferred cells.

To determine whether Ag-specific stimulation is required for in vivo expansion of Tr, PUVA-treated cells obtained from DNFB-sensitized donors were injected into naive recipients. In contrast to the experiment shown in Fig. 8, recipients were either left untreated or sensitized against OXA instead of DNFB. Five days after treatment spleen and lymph node cells were obtained and transferred i.v. into naive mice at a concentration of 5 x 107 or 1 x 105. 2° recipients were only suppressed upon injection of 5 x 107 cells, but not upon injection of 1 x 105 cells (Fig. 9). The same was observed when 1° recipients were left untreated as already shown in Fig. 8C. This implies that expansion of Tr in vivo only takes place upon Ag-specific boosting.



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  		FIGURE 9. In vivo expansion of Tr cells induced by 8-MOP/UVA is hapten-specific. Spleen and lymph node cells were obtained from mice that were sensitized against DNFB. Cells were exposed extracorporeally to 8-MOP plus UVA (PUVA). PUVA-treated cells were injected i.v. into naive syngeneic mice. Five days after injection, recipients were sensitized against OXA. Five days thereafter, lymph node cells were obtained from the recipient mice and transferred i.v. into naive 2° recipients. In addition, cells were obtained from 1° recipients, which received PUVA-treated cells but were not sensitized with DNFB (naive), and injected (5 x 107 or 1 x 105 cells) into naive 2° recipients. Twenty-four hours after transfer, mice were sensitized against DNFB. Ear challenge with DNFB was performed 5 days thereafter. Positive control mice were sensitized and challenged without transfer; negative control mice were ear challenged only. Ear swelling was measured 24 h after challenge. Ear swelling response is expressed as the difference (centimeters x 10–3, mean ± SD) between the thicknesses of the challenged ears compared with the thickness of the vehicle-treated ears.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The data indicate that the infusion of 8-MOP/UVA-treated leukocytes inhibits the immune response in an animal model of CHS via induction of Ag-specific regulation. The most convincing evidence of ECP clinical benefit exists for solid organ transplantation (28) and GvHD (29) with more limited reports in a variety of autoimmune and alloimmune disorders (30, 31, 32). It is intriguing to speculate that the mechanistic findings of this study explain these human results. Ongoing clinical trials are underway to determine whether this is indeed the case. We have confirmed the findings of others that ECP treatment induces the apoptotic cell death of virtually all leukocytes (33, 34). Neither 8-MOP nor UVA at this dose were capable of inducing significant cell death. CD3+ cells were >50% annexin V+ by day 1, while CD11c+ cell death was slightly delayed. We found that upon reinfusion, these preapoptotic cells are retained primarily in the spleen and liver with smaller amounts retained in the small intestine and secondary lymph nodes. It is presumed that in these sites they begin to express apoptotic-cell-associated molecular patterns and are removed from the body primarily by resident APC (21). There is growing evidence that clearance or scavenging of apoptotic cells by APC is not immunologically neutral, as once assumed. In animal models, it has now been clearly established that clearance of infused apoptotic cells in the absence of danger signals, irrespective of the cell type (25, 35, 36, 37, 38, 39, 40) or the means of apoptotic induction (23, 24, 41, 42, 43), induces immune tolerance (44, 45). It has been speculated that this immune modulation evolved as a system to constantly sample self Ag in the periphery and to inhibit immune autoreactivity (26). Thus, infusion of apoptotic cells may simply be a method to harness a natural process for therapeutic treatment of disease. The current view is that the apoptotic-cell-associated molecular pattern receptors on the surface of APCs recognize and engage apoptotic cells (21). This engagement leads to a series of events including phagocytic uptake of the apoptotic cells and signaling by a subset of these receptors. This signaling has been associated with a decreased ability to efficiently stimulate T effector cell responses (46), increased anti-inflammatory cytokine production (47, 48), decreased proinflammatory cytokine production (23, 35), and possibly aid the generation of Tr (11, 20).

Our data suggest that ECP-treated CD11c+ cells, which carry the hapten Ag, may have the ability to directly tolerize before their eventual demise. Using nonhaptenated CD11c+ cells or cells from animals sensitized 3 wk earlier did not induce immune tolerance (data not shown). In vitro we were able to demonstrate that human APCs treated with ECP induced the generation of CD4 cells with regulatory activity. This activity was lost when non-APC cells 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 circumstances such as this haptenated model it is essential that APCs carrying the Ag directly stimulate regulatory cells. In other situations, when the Ag is readily available in the host, the delivery of apoptotic cells may be disconnected from the Ag because they will meet in the secondary lymph node system. This latter system seems more likely the mechanism in diseases such as solid organ transplantation. What is clear is that the action of apoptotic cells is not direct at the site of inflammation because ECP-treated cells could not be detected in the inflamed ears.

For cutaneous UVB-induced immunomodulation there is good evidence that UVB-damaged cutaneous APC are involved in this process. When Langerhans cells are exposed to UVB they migrate into the draining lymph nodes and are not able to present the hapten properly, thereby failing to appropriately sensitize (49). There is evidence that this treatment directly stimulates regulatory cell generation, which is transferable to naive animals and suppresses future immune responses in an Ag-specific fashion (50). Reduction of DNA damage by exogenous repair enzymes restores the immunostimulatory capacity of APC obtained from lymph nodes draining UVB-exposed skin and the development of Tr is blocked upon administration of the DNA repair enzymes (51). IL-12, which is able to prevent UVB-induced immunosuppression (52), may work in part via its capacity to reduce UVB-mediated DNA damage (53, 54). Taken together, all these findings indicate that both the inhibition of the induction of CHS and the generation of Tr are a consequence of UVB-induced DNA damage in APC. Because, in contrast with the cutaneous UVB treatment, the 8-MOP/UVA treatment is ex vivo, it allows for an easier dissection of the system to determine the mechanism of action.

Although the functional characterization of clinical response to ECP is quite advanced and there is information on the downstream immune changes, the initiating events are not understood at all. In particular, we were interested which, if any, cell type might be targeted by 8-MOP/UVA. In this system we only observed immunomodulation by injecting 8-MOP/UVA-treated cells obtained from DNFB-sensitized donors, which led to the Ag specificity. We were quite surprised to see this activity even in cells obtained 5 days after sensitization. It was surmised that DNFB-specific T effector cells might be the primary target for 8-MOP/UVA, and that infusion of apoptotic DNFB-specific T effector cells might cause the induction of an anti-clonotypic T cell response, as has been suggested as a mechanism (7). However, when we depleted the spleen and lymph node cell suspension of Thy1.2+ cells before 8-MOP/UVA exposure, we still observed an inhibition in the immune response. When cells were depleted of CD11c+ cells, suppression was lost and suppression could not be transferred into the secondary recipient when treated splenocytes were depleted of CD11c+ cells, indicating that Tr had not developed in the 1° recipients (data not shown). This data implies that while all cells may undergo apoptosis following extracorporeal 8-MOP/UVA therapy, APCs are critically important in this system. In addition, suppression was only observed upon PUVA exposure of cells obtained from sensitized but not from naive mice. We propose that extracorporeal 8-MOP/UVA treatment of APC still carrying the hapten induces an apoptotic cascade, which may take place over several days. Infusion of these damaged, slowly dying APC does not induce sensitization but in contrast causes immunomodulation similar to the UVB-damaged Langerhans cells of the skin. Both the cutaneous UVB and the 8-MOP/UVA treated APC carry the hapten to the lymphoid organs. This hypothesis is supported by the observation that we do not induce suppression in the recipients when we inject PUVA-treated cells obtained from donors which had been sensitized 3 wk earlier (data not shown).

It is possible that in a nonhaptenated system, any apoptotic cell will induce tolerance, but in the case of haptens, the Ag must be linked on the apoptotic cell and only APC retain this ability following 5 days in vivo. This latter mechanism seems likely because apoptotic cells from pure T cells, cell lines, or even xenogeneic neutrophils have been shown to induce immune tolerance in other nonhapten-based systems. This suggests that, in contrast to the hapten system, treated APCs may not be critical for the induction of tolerance and a disconnect between the apoptotic cell and the tolerizing Ag is possible.

In human clinical trials immunophenotypic analysis of dendritic cell (DC) populations revealed a preponderance of DC1 monocytic DCs in all patients before the initiation of ECP (55). Nine of 10 patients demonstrated a shift from DC1 to DC2 and a concordant shift from a predominantly Th1 to Th2 cytokine profile after ECP, which would suggest that ECP alters alloreactivity by affecting allotargeted effector T cells and Ag-presenting DC. This study has been criticized because the definition for DC used was in contradiction to the standard classification of DC, the expansion was conducted ex vivo, and certain controls were missing (56). Nevertheless, this study and others favor the concept of the development of active specific rather than suppressive nonspecific immune responses induced by ECP (5, 57) the exact nature of which needs to be determined.

One of the primary mechanisms by which these tolerogenic APC may operate is via cytokine modulation. There is a large volume of literature from in vitro and some in vivo work showing apoptotic cell induction of anti-inflammatory cytokines and inhibition of proinflammatory cytokines by APC (reviewed in Ref. 21). This increase in anti-inflammatory and decrease in proinflammatory cytokines may, in part, be responsible for the clinical effects of ECP seen in humans and animals. The relative importance of these cytokine alterations vs the generation of regulatory cell activity that we describe in this study is still not clear.

In humans, evidence also suggests that ECP modulates cytokine secretion. There has been a reported decrease in proinflammatory cytokines TNF-{alpha} and IFN-{gamma} in CTCL and GvHD patients undergoing 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 IL-1 receptor antagonist. Gorgun et al. (55), using an ex vivo culture system, found an increase in IL-4 and IL-10. In vitro production of immunosuppressive cytokines, including IL-10 following ingestion of apoptotic cells, has been observed (48) and may be involved in the earlier therapeutic benefit reported in ECP (60). Others have also suggested systemic effects on leukocyte populations and function (55, 61). These data correspond to our ex vivo IL-10 production data reported in this study and our recent in vivo description showing a role for IL-10 in an UVB-induced Tr system (18).

It was shown >25 years ago that the topical application of contact allergens (haptens) does not induce sensitization when the hapten is applied onto UVB-exposed skin (62). In addition, individuals having received the allergen on UVB-exposed skin cannot be sensitized later against the very same hapten, implying that hapten-specific tolerance had developed (50). This tolerance is mediated, in part, via the induction of Ag-specific Tr, because suppression can be adoptively transferred into naive recipients (63). According to recent observations, UV-induced Tr belong to the CD4+/CD25+ subtype (18), express CTLA-4 (64), bind the lectin dectin-2 (63), and release IL-10 upon antigenic stimulation (64). Unfortunately, the complexity of the cutaneous system has confounded the mechanistic interpretation, and we are still left with an incomplete explanation of this phenomenon (65).

To get an idea whether ECP might also induce Tr, we set up an experimental in vivo model for ECP using CHS as a readout system. With this model we demonstrate that extracorporeal exposure of splenocytes and lymph node cells obtained from DNFB-sensitized donors to 8-MOP/UVA and subsequent infusion into naive mice inhibits sensitization against DNFB in the recipients. Immunomodulation is only observed when cells are subjected to a combination of 8-MOP and UVA, whereas each of these components alone does not. This immunomodulation is Ag-specific because the recipient mice respond normally to unrelated haptens. In addition, the PUVA-exposed cells have to be obtained from sensitized and not naive animals. In this system this effect is due to the induction of Tr because transfer of as few as 105 splenocytes from the recipients into another generation of naive mice renders the 2° recipients similarly unresponsive to DNFB. This is surprising considering we injected bulk cells obtained from 1° recipients, which presumably consists of only a small minority of hapten-specific Tr. It is presumed that in vivo expansion of Tr occurs because fewer cells suffice to transfer suppression when 1° recipients are sensitized with DNFB, presumably through increased in vivo proliferation or activity of the transferred cells. However, this in vivo proliferation and expansion requires Ag-specific stimulation because a higher number of cells was needed to be transferred to mediate suppression when donors were boosted instead with the specific Ag DNFB than with the nonspecific hapten OXA. Depletion studies revealed that 8-MOP/UVA-induced Tr are both CD4 and CD25 positive. They appear to be similar to UVB-induced Tr (18). We have not yet determined whether they express CTLA-4 or bind dectin-2. However, we do have in vitro evidence that 8-MOP/UVA-induced Tr release increased amounts of IL-10 and TGF-{beta} when coincubated with DC and the specific hapten.

Taken together, these results suggest that infusion of autologous haptenated cells in which apoptosis was induced by 8-MOP/UVA, 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-{beta}. 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 mechanism by which photopheresis may induce immunological tolerance will determine ways to better use the current therapy and indicate ways in which improvements can be made for the benefit of our patients.


   Acknowledgments
 
We are grateful to Eva Emmell and Terry Goletz for conducting the radiolabelled cell tracking study and Stefan Beissert for critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
D. Peritt is an employee of, and T. Schwarz received grant support from, Therakos, a Johnson & Johnson company.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the German Research Foundation (Sonderforschungsbereich 293, B9 to T.S.; SCHW1177/1-1 to A.S and T.S.) and a grant from Therakos (to T.S.). Back

2 Address correspondence and reprint requests to Dr. Thomas Schwarz, Department of Dermatology, University Kiel, Schittenhelmstrasse 7, D-24105 Kiel, Germany. E-mail address: tschwarz{at}dermatology.uni-kiel.de Back

3 Abbreviations used in this paper: PUVA, psoralen plus UVA; CHS, contact hypersensitivity; CTCL, cutaneous T cell lymphoma; DC, dendritic cell; DNFB, 2,4-dinitrofluorobenzene; ECP, extracorporeal photopheresis; GvHD, graft-vs-host disease; 8-MOP, 8-methoxypsoralen; OXA, oxazolone; Tr, regulatory T cell. Back

Received for publication October 14, 2004. Accepted for publication February 27, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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