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Extracorporeal photochemotherapy does not suppress T- or B-cell responses to novel or recall antigens
Extracorporeal photochemotherapy does not suppress T- or B-cell responses to novel or recall antigens Karen Rebecca Suchin, MD, Maureen Cassin, BS, Rita Washko, MD, George Nahass, MD, Michael Berkson, MD, Bruce Stouch, MS, Benjamin R. Vowels, PhD,† and Alain H. Rook, MD Philadelphia, Pennsylvania Background: Extracorporeal photopheresis (ExP) is an effective therapy for several conditions including cutaneous T-cell lymphoma, scleroderma, and allograft rejection. Experimental animal models suggest that ExP may induce antigen-specific immunosuppression. Objective: Our purpose was to determine the effect of photopheresis on humoral and cell-mediated immunity in human subjects. Methods: Recall and primary immune responses of patients with scleroderma receiving monthly ExP treatments were assessed by delayed type hypersensitivity skin tests, T-cell proliferative responses after immunizations with tetanus toxoid and keyhole limpet hemocyanin, and serum antibody titers against common viral pathogens. Results: After 6 months of ExP, viral antibody titers and delayed type hypersensitivity responses were not significantly different from baseline values in all 7 patients tested. T-cell responses to tetanus toxoid remained normal in 3 of 3 patients tested for a minimum of 6 months after booster immunization. Immunization with the protein antigen keyhole limpet hemocyanin after initiation of ExP therapy resulted in sustained T-cell proliferative responses up to 6 months in 3 of 3 patients. Conclusion: These results, along with the observation of no increased incidence of opportunistic infections or neoplasms, suggest that ExP is not broadly immunosuppressive and does not prevent primary responses to vaccination or other antigenic challenges. (J Am Acad Dermatol 1999;41:980-6.)
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hotopheresis is a form of apheresis-based immunomodulatory therapy in which peripheral blood leukocytes are exposed to UVA radiation extracorporeally 2 hours after the ingestion of 8-methoxypsoralen (8-MOP). After exposure to UVA, the leukocytes are returned to the patient. Results of recent multicenter trials have demonstrated that patients with advanced forms of cutaneous T-cell lymphoma (CTCL)1,2 and progressive systemic sclerosis (scleroderma)3 can derive substantial benefit from extracorporeal photopheresis (ExP). Furthermore, a controlled trial has demonstrated that From the Department of Dermatology, School of Medicine, University of Pennsylvania. Accepted for publication June 24, 1999. Reprint requests: Alain Rook, MD, Department of Dermatology, Hospital of the University of Pennsylvania, 2 Rhoads Pavilion, 3600 Spruce St, Philadelphia, PA 19104-4283. †Deceased. Copyright © 1999 by the American Academy of Dermatology, Inc. 0190-9622/99/$8.00 + 0 16/1/100898
980
photopheresis can be successfully used to prevent cardiac allograft rejection,4 and a recent large series suggests that photopheresis is effective therapy for chronic graft-versus-host disease (GVHD).5 The mechanism by which ExP is able to effect these changes remains unclear; however, recent data suggest that cytokines, induced during the processing of the leukocytes, contribute to the overall effectiveness of ExP.6 Because exposure of leukocytes, and in particular T lymphocytes, to UVA subsequent to uptake of 8-MOP results in the alteration and ultimate destruction of these cells,7,8 it is possible that a generalized immunosuppression occurs in these patients. This possibility is supported by the observation that patients undergoing long-term psoralen + UVA (PUVA) therapy for the treatment of psoriasis have impaired T-cell function.9 The effectiveness of 8-MOP and UVA to suppress allograft rejection10 and to control autoimmune disease11,12 and GVHD13 in humans and experimental animal mod-
J AM ACAD DERMATOL VOLUME 41, NUMBER 6
els would also appear to support an immunosuppressive mechanism. To investigate this possibility, we tested T- and Bcell function in a group of patients with CTCL or systemic sclerosis undergoing ExP. In particular, B-cell function was assessed by measurements of total serum immunoglobulins (IgG) as well as serum levels of antibodies to specific viral antigens and to a novel protein antigen, keyhole limpet hemocyanin (KLH), after primary challenge. To assess T-cell function, we examined in vivo delayed-type hypersensitivity (DTH) reactions and proliferative responses to a recall antigen, tetanus toxoid (TT). As an important additional parameter of T-cell function we examined the ability of patients currently undergoing ExP to develop a primary de novo immune response after vaccination with the protein KLH.
MATERIAL AND METHODS Patients Seven patients with recent-onset systemic sclerosis of less than 3 years’ duration who were to begin receiving ExP were selected for this study. In addition, 2 patients with CTCL, who were also receiving ExP, were included in antigen response studies. The ExP regimen consisted of treatments on 2 consecutive days every 4 weeks as previously described.3 Patients evaluated in this study had no previous or concurrent history of immunosuppressive disease or corticosteroid drug therapy and no evidence of acute infection. This study conformed to institutional review board–approved protocols and informed consent was obtained before sample acquisition.
Study design Baseline measurements of all tested parameters except response to primary antigen challenge were performed before initiation of ExP. Follow-up measurements of total serum immunoglobulin levels, viral antibody titers, and DTH skin responses were performed after patients had received 6 months of ExP. To assess response to primary or recall antigen challenge, patients undergoing ExP were subcutaneously immunized with 5 mg of KLH (a generous gift of Dr H. Clifford Lane, National Institute of Allergy and Infectious Diseases, National Institutes of Health) or 0.5 mL of TT (Wyeth Laboratories, Radnor, Pa) immediately after receiving a course of ExP. Two weeks later, the patients received an additional 5 mg of KLH. Three normal controls were also vaccinated with KLH and their cellular immune responses to this novel antigen were assessed.
Serum immunoglobulin level assessment Quantitative total serum immunoglobulins were measured by means of the SPQ TM Test System for Serum Proteins by turbidimetric analysis (Atlantic Antibodies, Inc, Scarborough, Me). The normal range of values for total serum IgG is 650 to 1900 mg/dL for males and 650 to 2000 mg/dL for females.
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Assessment of antibodies to recall antigens As a measure of antibody response to antigens to which the patients had been previously exposed, commercially available enzyme-linked immunosorbent assay (ELISA) kits (Whittaker Bioproducts, Walkersville, Md) were used to determine the level of antibodies against varicella zoster virus, cytomegalovirus (CMV), and Epstein-Barr virus.
Antibody response to recall and primary challenge antigens To determine the levels of antibody against KLH after primary challenge or to the recall antigen TT, 96-well flatbottom plates were coated with 1 µg KLH or TT per well in a polycarbonate buffer (pH 9.6) and incubated at 4°C for 16 hours. The plates were then washed with PBS/Tween 20, incubated with blocking solution (1% nonfat dry milk in PBS, pH 7.2), and washed again. Next, 200 µL of a 1:10,000 dilution of patient’s serum was added to each well. The plate was incubated at 37°C for 16 hours and then washed with PBS/Tween 20. Peroxidase-labeled goat anti-human IgG F(ab’)2 (Lampire Biological Laboratories, Pipersville, Pa) was diluted 1:100,000, added in a volume of 200 µL/well, and incubated for 2.5 hours at 37°C. The plates were then washed and incubated with 100 µL of the substrate o-phenylenediamine dihydrochloride (OPD, Sigma, St Louis, Mo) for 20 minutes in the dark at room temperature. After this incubation, 50 µL of 2NH2SO4 was added and the O.D. 492 for each well was determined.
Delayed-type hypersensitivity skin testing DTH skin reactivity to an intradermal injection of 0.1 mL of the following reagents was determined: (1) PPD (Parke-Davis, Morris Plains, NJ), (2) Dermatophyte (Dilution 1:30, Hollister Stier Miles, Inc, Elkhart, Ind), (3) Mumps Skin Test Antigen (Connaught Laboratories, Swiftwater, Pa), (4) Candida albicans (Greer Laboratories, Lenou, NC), and (5) Tetanus Toxoid Fluid (Lederle Labs, Pearl River, NY). Reactivity was measured in millimeters of induration at 48 hours after the anergy panel was placed. A change of more than 5 mm from baseline was considered clinically relevant for our panel of antigens.14
In vitro T-cell proliferation assays Peripheral blood mononuclear cells, isolated from a Ficoll-Hypaque (Ficoll-Paque, Pharmacia, Piscataway, NJ) gradient preparation of heparinized whole blood, were plated in triplicate in a 96-well flat-bottom plate (Corning Glass Works, Corning, NY) at a concentration of 5 × 105 cells/mL in a final volume of 200 µL of RPMI 1640 (JHR Biosciences, Lenexa, Kan) supplemented with 10% FCS (JHR), L-glutamine, penicillin, and streptomycin (Sigma Chemical, St Louis, Mo). TT (10 to 50 µg/mL) or KLH (10 to 100 µ/mL) was added to the assay wells, and the plate was incubated at 37°C with 5% CO2 for 5 days whereupon 1 µCi of 3H-thymidine (NEN, Boston, Mass) was added to each well and the plate incubated an additional 16 hours. The wells were then harvested onto glass fiber filters and the radioactivity of each well determined on a scintillation counter (Packard Tri-Carb 300, Sterling, Va). Antigen
982 Suchin et al
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Fig 1. Total serum immunoglobulin G levels before and after 6 months of extracorporeal photopheresis. Normal range of total serum IgG is 650 to 1900 mg/dL for males and 650 to 2000 mg/dL for females.
A
Table I. Effect of extracorporeal photopheresis on antibody titers to viral antigens Patient No.
2
4
5
B
Antigen
Baseline O.D.*
Posttreatment O.D.
∆O.D.†
VZV HSV CMV EBV VZV HSV CMV EBV VZV HSV CMV EBV
3.52 5.32 3.31 NT† 2.49 5.17 1.02 ‡ 4.70 2.61 6.16 ‡
3.32 5.02 2.74 NT 2.80 5.66 1.44 ‡ 5.13 2.85 6.57 ‡
–0.2 –0.3 –0.57 — +0.31 +0.49 +0.42 ‡ +0.43 +0.24 +0.41 ‡
CMV, Cytomegalovirus; EBV, Epstein-Barr virus; HSV, herpes simplex virus; NT, not tested; VZV, varicella zoster virus. *Antibody titer determined by ELISA. †∆O.D. was not significant after treatment in any of the patients tested. ‡EBV titers to viral capsid, nuclear, and early antigens were examined and no significant difference in the ∆O.D. was observed.
Fig 2. Antibody response to recall and novel antigens in patients receiving extracorporeal photopheresis. Patients’ sera were tested 2 months after booster with antigen and demonstrate a significant antibody response to both TT and KLH. This response was maintained for at least 6 months (data not shown). A, Response to TT. B, Response to KLH.
response was assessed by a stimulation index (SI) that was calculated by means of the formula SI=experimental cpm/control cpm, where control cpm is the spontaneous proliferation of cells not exposed to antigen. An SI of more than 2.0 was considered a significant response.
Statistical analysis Analysis of DTH data was performed by means of the
Wilcoxon signed rank test or the paired difference t test. A P value of less than .05 was considered statistically significant.
RESULTS Effect of ExP on humoral immunity To assess the overall effects of ExP on humoral immunity, titers (in milligrams per deciliter) of serum immunoglobulins (IgG, IgA, and IgM) were determined before the initiation of therapy and after 6 months of therapy as described above. Six of 7 patients had total serum IgG levels within the normal range; 1 patient had elevated levels of circulating IgG. Total serum IgG levels were unchanged after 6 months of photopheresis therapy in all 7 patients (Fig 1). In addition, no changes were observed in the IgA or IgM
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Table II. Effect of extracorporeal photopheresis on cutaneous delayed-type hypersensitivity reaction to common antigens Antigen* Patient No.
1 2 3 4 5 6 7 Summary
Sample†
PPD
Trichophyton
Mumps
Candida
Tetanus
Baseline Post Baseline Post Baseline Post Baseline Post Baseline Post Baseline Post Baseline Post Median ∆‡ Paired difference§
0 0 25 8 0 0 0 0 0 0 0 0 5 0 0 P > .5
0 5 10 6 0 0 0 0 0 0 0 5 0 0 0 P > .5
0 5 0 0 5 6 8 8 0 3 10 5 0 0 0 P > .7
15 7 0 0 0 7 8 10 5 10 15 8 0 0 0 P > .8
10 8 8 0 0 0 10 0 10 0 0 2 8 5 –3.0 P > .09
*Response represented as millimeters of induration measured 48 hours postchallenge. †Baseline data obtained before initiation of photopheresis therapy; posttherapy data obtained after 6 months of photopheresis therapy. ‡Change in millimeters of induration between baseline and post-therapy measurements. §Tested by Wilcoxon signed rank test; a change of ≥ 5.0 mm from baseline was considered clinically relevant.
titers (data not shown). Furthermore, antibody titers against CMV, varicella zoster virus, and Epstein-Barr virus were not significantly changed after 6 months of therapy (Table I). Thus neither nonspecific nor specific immunoglobulin levels appeared to be altered by a 6-month course of photopheresis therapy. Antibody responses to a recall antigen, TT, were enhanced and sustained after a booster for the duration of this study. A high level of antibody response to primary antigenic challenge with KLH was observed in 3 of 3 patients tested (Fig 2). This level of response was maintained over the course of this study (minimum 6 months). Effect of ExP on cell-mediated immunity To evaluate whether ExP suppresses cell-mediated immunity, DTH skin test responses to a panel of common antigens were assessed in systemic sclerosis patients receiving this therapy. All 7 patients tested just before initiation of photopheresis displayed a positive skin test, consisting of 5 mm or more of cutaneous induration to 1 or more of the antigens (Table II). Skin test reactivity was studied after 6 months of ExP, and a change of 5 mm or more in induration from baseline was considered significant. By this criterion, 3 of the subjects who had a pretherapy response to tetanus completely lost their ability to respond to this antigen after 6 months of photopheresis. However,
there was no significant difference in the cutaneous DTH responses of the overall patient population to the test panel as determined by either the Wilcoxon signed rank test or the paired difference t test. To further determine whether ExP altered the patients’ T-cell–mediated immunity to a recall antigen, we examined the in vitro response to TT. Because administration of a booster dose of TT to patients originally enrolled in the study could interfere with the assessment of cutaneous DTH responses to TT after ExP, thus invalidating the results, 3 additional patients undergoing ExP, 2 with CTCL, received a TT booster; T-cell proliferation to the antigen was assessed in vitro at a minimum of 3 separate intervals. A significant proliferative response to TT was observed in all 3 patients tested at 1 month after booster (Table III). This response was maintained for at least 6 months in all 3 patients tested. Because it is possible that cell-mediated recall responses are less sensitive to the possible suppressive effects of ExP than are primary cell-mediated responses, we examined the patients’ ability to generate a cell-mediated immune response to KLH after primary challenge. In all 3 patients tested, a significant in vitro proliferative response was observed 1 month after challenge (Table III). This response was maintained over a period of at least 6 months in all 3 patients tested.
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Table III. In vitro T-cell proliferation responses to recall and de novo antigens in patients receiving extracorporeal photopheresis Antigen* Patient
A
B
C
Control 1
Control 2
Assay date†
Tetanus toxoid‡
KLH§
Baseline 1 mo 2 mo 3 mo 4 mo 11 mo Baseline 1 mo 8 mo 10 mo Baseline 1 mo 4 mo 6 mo 1 mo 2 mo 3 mo 1 mo 2 mo 3 mo
1.5 6.7 2.7 NT 2.0 4.5 1.0 6.3 1.0 2.2 1.0 3.7 3.0 2.0 5.3 2.4 2.8 5.8 3.3 2.9
1.0 54.1 2.1 3.3 3.4 9.7 1.0 5.3 2.2 1.0 1.0 2.7 3.3 3.2 4.2 3.3 5.8 2.8 4.7 5.0
NT, Not tested. *Response represented as stimulation index (SI) = experimental cpm/control cpm. An SI of ≥ 2.0 was considered significant. Each sample condition was assayed in triplicate. †Months after immunization. Monthly samples for assay were obtained before initiation of that month’s ExP therapy. ‡Responses to tetanus toxoid (TT) were assayed at in vitro concentrations of 10 to 50 µg/mL. Patients, but not controls received TT booster at baseline. §Patients and controls received a 5 mg immunization and subsequent 5 mg booster at 2 weeks. Responses to KLH were assayed at in vitro concentrations of 10 to 100 µg/mL.
DISCUSSION ExP has been demonstrated to be an effective therapeutic modality in the treatment of CTCL,1 for several autoimmune diseases including systemic sclerosis and pemphigus vulgaris,3,11,12 acute and chronic GVHD,5 and in the prevention of acute cellular rejection in recipients of cardiac transplants.4 Although the precise mechanism of action has not been determined, exposure of leukocytes to UVA after uptake of 8-MOP results in cross-linking of DNA and eventual apoptotic cell death.15,16 Murine skin transplantation models indicate that the treated leukocytes are altered in some fashion such that, when reinfused, a specific immunosuppressive response directed towards the treated cells occurs.10 On the basis of these observations, it would appear
that a generalized immunosuppression could occur as a result of this therapy either via induction of an immunosuppressive response or through the nonspecific loss of leukocytes. If such an effect does indeed occur, it would warrant exclusion of certain types of patients from this form of therapy to prevent exacerbation of disease via secondary infections or neoplasms. Furthermore, a generalized immunosuppression would preclude the use of various vaccines, particularly those with attenuated organisms, which are also important in maintaining the health of the patient. The present study indicates, however, that ExP does not induce a generalized immunosuppression in either the cellular or humoral component of the immune system. Our study demonstrates that patients undergoing ExP for either the treatment of scleroderma or CTCL are able to generate both Tand B-cell responses to both recall and novel antigens as evidenced by results of numerous immunologic assays. The results of this study are in contrast to those of Vonderheid et al,17 who reported that 4 of 4 patients with psoriasis who underwent photopheresis treatments lost their cutaneous DTH response and the in vitro capacity of their mononuclear cells to produce IL-2 in response to mitogens. However, the patients with psoriasis were pretreated with or received concomitant methotrexate therapy. One patient also received systemic corticosteroids along with ExP. In these patients, the generalized immunosuppression was likely because of the administration of methotrexate and corticosteroids. Quantification of serum immunoglobulin levels revealed that ExP did not suppress general or specific B-cell function. Baseline and follow-up measurements of total and specific recall Ig levels remained unchanged. When the more sensitive ELISA was used to measure secretion of antigen-specific immunoglobulins to TT and KLH, the patients universally demonstrated the ability to generate antibody responses to both the recall and novel antigens after undergoing multiple cycles of photopheresis. Results of the DTH testing showed that all patients retained cutaneous reactivity against one or more antigens. Although there appeared to be some loss of the response to specific antigens such as TT and PPD in individual patients, there was no statistically significant variation in response to any of the tested antigens among the total patient population. Nevertheless, we cannot unequivocally conclude that photopheresis does not suppress tetanus-specific responses. The ability of the patients to generate recall and primary cellular immune responses to specific antigens as assessed both in vivo (cutaneous DTH) and in vitro (T-cell proliferation assays) indi-
J AM ACAD DERMATOL VOLUME 41, NUMBER 6
cates that patients receiving ExP are not inherently more vulnerable to opportunistic or other infectious agents. Indeed, in our experience, which includes treatment of more than 125 patients in a 12-year period, there have been no incidents of opportunistic infections or neoplasms as a result of this therapy. One indicator of general immunosuppression is the reactivation of latent herpesvirus infection, which frequently occurs in the setting of the extended administration of immunosuppressive drugs. Increased antibody titers to herpesviruses suggests reactivation of latent infection and therefore poor cellular immune control of latent virus. The failure to observe a specific rise in serum antibodies against any of the herpesviruses in our study is further evidence that photopheresis does not induce a profound T-cell immunosuppression. Moreover, in a recent multicenter controlled trial using photopheresis to prevent cardiac allograft rejection, the incidence of CMV reactivation was lower in the photopheresis group than in the control group.4 Evidence that photopheresis induces specific immunity against pathogenic T-cell clones has been supported by recent observations using experimental animal models. Study of the murine model of experimental allergic encephalitis by Khavari et al18 has been especially suggestive in this regard. In this model, rats injected with myelin basic protein experience a paralytic illness associated with T-cell destruction of the nervous system. The pathogenic T cells that mediate this destruction can be isolated and cloned in vitro. When naive syngeneic rats are injected with the cloned T cells, all of the features of experimental allergic encephalitis are reproduced. However, if the pathogenic clones are first treated with psoralen and UVA and then infused, the animals are protected from the onset of disease upon subsequent challenge with the pathogenic T cells. Protection from disease appears to be mediated by the generation of clone-specific suppressor T cells that have developed in response to the psoralen and UVA-modified pathogenic cells. Similarly, Perez et al,19 using a model of cutaneous allograft rejection, have also obtained evidence of stimulation of an antigen-specific suppressor T-cell response when alloreactive effector T cells are treated with psoralen and UVA and infused into syngeneic animals.19 These results indicate that, at least in the setting of these animal models, an active immunization process can occur after administration of photoinactivated syngeneic T-cell clones and that generalized immunosuppression does not occur. Thus, the induction of anticlonotypic immunity is the presumed mechanism of action of photopheresis in the treatment of a variety of patholog-
Suchin et al 985
ic conditions including CTCL, allograft rejection, and GVHD. Although the mechanism by which ExP exerts its therapeutic effects is not wholly appreciated, these data demonstrate that its efficacy is not a result of generalized immunosuppression. This may explain the observation that patients undergoing ExP have no greater risk of experiencing opportunistic infections or neoplasms compared with the normal population. In addition, it suggests that attenuated live vaccines may be used in the preventive health management of patients receiving photopheresis. Based on our results we recommend administration of vaccines at the conclusion of a 2-day treatment cycle. REFERENCES 1. Edelson R, Berger C, Gasparro F, Jegasothy B, Heald P, Wintroub B, et al. Treatment of cutaneous T-cell lymphoma by extracorporeal photochemotherapy: preliminary results. N Engl J Med 1987;316:297-303. 2. Gottleib SL, Wolfe JT, Fox FE, DeNardo BJ, Macey WH, Bromley PG, et al. Treatment of cutaneous T-cell lymphoma with extracorporeal photopheresis monotherapy and in combination with recombinant interferon alpha: a 10-year experience at a single institution. J Am Acad Dermatol 1996;35:946-57. 3. Rook AH, Freundlich B, Jegasothy BV, Perez MI, Barr WG, Jimenez SA, et al. Treatment of systemic sclerosis with extracorporeal photochemotherapy: results of a multicenter trial. Arch Dermatol 1992;128:337-46. 4. Barr ML, Meiser BM, Eisen HJ, Roberts RF, Livi U, Dall’Amico R, et al. Photopheresis for the prevention of rejection in cardiac transplantation. N Engl J Med 1998;339:1744-50. 5. Greinix HT, Volc-Platzer B, Rabitsch W, Gmeinhart B, GuevaraPineda C, Kalhs P, et al. Successful use of extracorporeal photochemotherapy in the treatment of severe acute and chronic graft-versus-host disease. Blood 1998;92:3098-104. 6. Vowels BR, Cassin M, Boufal MH, Walsh LJ, Rook AH. Extracorporeal photochemotherapy induces the production of tumor necrosis factor-alpha by monocytes: implications for the treatment of cutaneous T-cell lymphoma and systemic sclerosis. J Invest Dermatol 1992;98:686-92. 7. Kraemer KH, Waters HL, Cohen LF, Popescu NC, Amsbaugh SC, DiPaolo JA, et al. Effects of 8-methoxypsoralen and ultraviolet radiation on human lymphoid cells in vitro. J Invest Dermatol 1981;76:80-7. 8. Berger CL, Cantor C, Welsh J, Dervan P, Begley T, Grant S, et al. Comparison of synthetic psoralen derivatives and 8-MOP in the inhibition of lymphocyte proliferation. Ann NY Acad Sci 1985; 453:80-90. 9. Morison WL, Wimberly J, Parrish JA, Bloch KJ. Abnormal lymphocyte function following long-term PUVA therapy for psoriasis. Br J Dermatol 1983;108:445-50. 10. Perez M, Edelson R, Laroche L, Berger C. Inhibition of antiskin allograft immunity by infusions with syngeneic photoinactivated effector lymphocytes. J Invest Dermatol 1989;92:669-76. 11. Rook AH, Freundlich B, Nahass GT,Washko R, Macelis B, Skolnicki M, et al. Treatment of autoimmune disease with extracorporeal photochemotherapy: progressive systemic sclerosis. Yale J Biol Med 1989;62:639-45. 12. Rook AH. Photopheresis in the treatment of autoimmune disease: experience with pemphigus vulgaris and systemic sclerosis. Ann NY Acad Sci 1991;636:209-16. 13. Ullrich SE. Photoinactivation of T-cell function with psoralen
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and UVA radiation suppresses the induction of experimental murine graft-versus-host disease across major histocompatibility barriers. J Invest Dermatol 1991;96:303-8. 14. Stallone DD, Stunkard AJ, Zweiman B, Wadden TA, Foster GD. Decline in delayed type hypersensitivity response in obese women following weight reduction. Clin Diagnostic Lab Immunol 1994;1:202-5. 15. Gasparro FP, Dall’Amico R, Goldminz D, Simmons E, Weingold D. Molecular aspects of extracorporeal photochemotherapy. Yale J Biol Med 1989;62:579-93. 16. Yoo EK, Rook AH, Elenitsas R, Gasparro FP, Vowels BR. Apoptosis induction by ultraviolet light A and photochemotherapy in
O
cutaneous T-cell lymphoma: relevance to mechanism of therapeutic action. J Invest Dermatol 1996;107:235-42. 17. Vonderheid EC, Bigler RD, Rogers TJ, Kadin ME, Griffin TD. Effect of extracorporeal photopheresis on selected immunologic parameters in psoriasis vulgaris.Yale J Biol Med 1989;62:653-64. 18. Khavari PA, Edelson RL, Lider O, Gasparro FP, Weiner HL, Cohen IR. Specific vaccination against photoinactivated cloned T-cells. Clin Res 1988;36:662. 19. Perez M, Edelson R, LaRoche L, Berger C. Specific suppression of antiallograft immunity by immunization with syngeneic photoinactivated effector lymphocytes. J Invest Dermatol 1989;92: 669-76.
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P
hotopheresis is a form of apheresis-based immunomodulatory therapy in which peripheral blood leukocytes are exposed to UVA radiation extracorporeally 2 hours after the ingestion of 8-methoxypsoralen (8-MOP). After exposure to UVA, the leukocytes are returned to the patient. Results of recent multicenter trials have demonstrated that patients with advanced forms of cutaneous T-cell lymphoma (CTCL)1,2 and progressive systemic sclerosis (scleroderma)3 can derive substantial benefit from extracorporeal photopheresis (ExP). Furthermore, a controlled trial has demonstrated that From the Department of Dermatology, School of Medicine, University of Pennsylvania. Accepted for publication June 24, 1999. Reprint requests: Alain Rook, MD, Department of Dermatology, Hospital of the University of Pennsylvania, 2 Rhoads Pavilion, 3600 Spruce St, Philadelphia, PA 19104-4283. †Deceased. Copyright © 1999 by the American Academy of Dermatology, Inc. 0190-9622/99/$8.00 + 0 16/1/100898
980
photopheresis can be successfully used to prevent cardiac allograft rejection,4 and a recent large series suggests that photopheresis is effective therapy for chronic graft-versus-host disease (GVHD).5 The mechanism by which ExP is able to effect these changes remains unclear; however, recent data suggest that cytokines, induced during the processing of the leukocytes, contribute to the overall effectiveness of ExP.6 Because exposure of leukocytes, and in particular T lymphocytes, to UVA subsequent to uptake of 8-MOP results in the alteration and ultimate destruction of these cells,7,8 it is possible that a generalized immunosuppression occurs in these patients. This possibility is supported by the observation that patients undergoing long-term psoralen + UVA (PUVA) therapy for the treatment of psoriasis have impaired T-cell function.9 The effectiveness of 8-MOP and UVA to suppress allograft rejection10 and to control autoimmune disease11,12 and GVHD13 in humans and experimental animal mod-
J AM ACAD DERMATOL VOLUME 41, NUMBER 6
els would also appear to support an immunosuppressive mechanism. To investigate this possibility, we tested T- and Bcell function in a group of patients with CTCL or systemic sclerosis undergoing ExP. In particular, B-cell function was assessed by measurements of total serum immunoglobulins (IgG) as well as serum levels of antibodies to specific viral antigens and to a novel protein antigen, keyhole limpet hemocyanin (KLH), after primary challenge. To assess T-cell function, we examined in vivo delayed-type hypersensitivity (DTH) reactions and proliferative responses to a recall antigen, tetanus toxoid (TT). As an important additional parameter of T-cell function we examined the ability of patients currently undergoing ExP to develop a primary de novo immune response after vaccination with the protein KLH.
MATERIAL AND METHODS Patients Seven patients with recent-onset systemic sclerosis of less than 3 years’ duration who were to begin receiving ExP were selected for this study. In addition, 2 patients with CTCL, who were also receiving ExP, were included in antigen response studies. The ExP regimen consisted of treatments on 2 consecutive days every 4 weeks as previously described.3 Patients evaluated in this study had no previous or concurrent history of immunosuppressive disease or corticosteroid drug therapy and no evidence of acute infection. This study conformed to institutional review board–approved protocols and informed consent was obtained before sample acquisition.
Study design Baseline measurements of all tested parameters except response to primary antigen challenge were performed before initiation of ExP. Follow-up measurements of total serum immunoglobulin levels, viral antibody titers, and DTH skin responses were performed after patients had received 6 months of ExP. To assess response to primary or recall antigen challenge, patients undergoing ExP were subcutaneously immunized with 5 mg of KLH (a generous gift of Dr H. Clifford Lane, National Institute of Allergy and Infectious Diseases, National Institutes of Health) or 0.5 mL of TT (Wyeth Laboratories, Radnor, Pa) immediately after receiving a course of ExP. Two weeks later, the patients received an additional 5 mg of KLH. Three normal controls were also vaccinated with KLH and their cellular immune responses to this novel antigen were assessed.
Serum immunoglobulin level assessment Quantitative total serum immunoglobulins were measured by means of the SPQ TM Test System for Serum Proteins by turbidimetric analysis (Atlantic Antibodies, Inc, Scarborough, Me). The normal range of values for total serum IgG is 650 to 1900 mg/dL for males and 650 to 2000 mg/dL for females.
Suchin et al 981
Assessment of antibodies to recall antigens As a measure of antibody response to antigens to which the patients had been previously exposed, commercially available enzyme-linked immunosorbent assay (ELISA) kits (Whittaker Bioproducts, Walkersville, Md) were used to determine the level of antibodies against varicella zoster virus, cytomegalovirus (CMV), and Epstein-Barr virus.
Antibody response to recall and primary challenge antigens To determine the levels of antibody against KLH after primary challenge or to the recall antigen TT, 96-well flatbottom plates were coated with 1 µg KLH or TT per well in a polycarbonate buffer (pH 9.6) and incubated at 4°C for 16 hours. The plates were then washed with PBS/Tween 20, incubated with blocking solution (1% nonfat dry milk in PBS, pH 7.2), and washed again. Next, 200 µL of a 1:10,000 dilution of patient’s serum was added to each well. The plate was incubated at 37°C for 16 hours and then washed with PBS/Tween 20. Peroxidase-labeled goat anti-human IgG F(ab’)2 (Lampire Biological Laboratories, Pipersville, Pa) was diluted 1:100,000, added in a volume of 200 µL/well, and incubated for 2.5 hours at 37°C. The plates were then washed and incubated with 100 µL of the substrate o-phenylenediamine dihydrochloride (OPD, Sigma, St Louis, Mo) for 20 minutes in the dark at room temperature. After this incubation, 50 µL of 2NH2SO4 was added and the O.D. 492 for each well was determined.
Delayed-type hypersensitivity skin testing DTH skin reactivity to an intradermal injection of 0.1 mL of the following reagents was determined: (1) PPD (Parke-Davis, Morris Plains, NJ), (2) Dermatophyte (Dilution 1:30, Hollister Stier Miles, Inc, Elkhart, Ind), (3) Mumps Skin Test Antigen (Connaught Laboratories, Swiftwater, Pa), (4) Candida albicans (Greer Laboratories, Lenou, NC), and (5) Tetanus Toxoid Fluid (Lederle Labs, Pearl River, NY). Reactivity was measured in millimeters of induration at 48 hours after the anergy panel was placed. A change of more than 5 mm from baseline was considered clinically relevant for our panel of antigens.14
In vitro T-cell proliferation assays Peripheral blood mononuclear cells, isolated from a Ficoll-Hypaque (Ficoll-Paque, Pharmacia, Piscataway, NJ) gradient preparation of heparinized whole blood, were plated in triplicate in a 96-well flat-bottom plate (Corning Glass Works, Corning, NY) at a concentration of 5 × 105 cells/mL in a final volume of 200 µL of RPMI 1640 (JHR Biosciences, Lenexa, Kan) supplemented with 10% FCS (JHR), L-glutamine, penicillin, and streptomycin (Sigma Chemical, St Louis, Mo). TT (10 to 50 µg/mL) or KLH (10 to 100 µ/mL) was added to the assay wells, and the plate was incubated at 37°C with 5% CO2 for 5 days whereupon 1 µCi of 3H-thymidine (NEN, Boston, Mass) was added to each well and the plate incubated an additional 16 hours. The wells were then harvested onto glass fiber filters and the radioactivity of each well determined on a scintillation counter (Packard Tri-Carb 300, Sterling, Va). Antigen
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J AM ACAD DERMATOL DECEMBER 1999
Fig 1. Total serum immunoglobulin G levels before and after 6 months of extracorporeal photopheresis. Normal range of total serum IgG is 650 to 1900 mg/dL for males and 650 to 2000 mg/dL for females.
A
Table I. Effect of extracorporeal photopheresis on antibody titers to viral antigens Patient No.
2
4
5
B
Antigen
Baseline O.D.*
Posttreatment O.D.
∆O.D.†
VZV HSV CMV EBV VZV HSV CMV EBV VZV HSV CMV EBV
3.52 5.32 3.31 NT† 2.49 5.17 1.02 ‡ 4.70 2.61 6.16 ‡
3.32 5.02 2.74 NT 2.80 5.66 1.44 ‡ 5.13 2.85 6.57 ‡
–0.2 –0.3 –0.57 — +0.31 +0.49 +0.42 ‡ +0.43 +0.24 +0.41 ‡
CMV, Cytomegalovirus; EBV, Epstein-Barr virus; HSV, herpes simplex virus; NT, not tested; VZV, varicella zoster virus. *Antibody titer determined by ELISA. †∆O.D. was not significant after treatment in any of the patients tested. ‡EBV titers to viral capsid, nuclear, and early antigens were examined and no significant difference in the ∆O.D. was observed.
Fig 2. Antibody response to recall and novel antigens in patients receiving extracorporeal photopheresis. Patients’ sera were tested 2 months after booster with antigen and demonstrate a significant antibody response to both TT and KLH. This response was maintained for at least 6 months (data not shown). A, Response to TT. B, Response to KLH.
response was assessed by a stimulation index (SI) that was calculated by means of the formula SI=experimental cpm/control cpm, where control cpm is the spontaneous proliferation of cells not exposed to antigen. An SI of more than 2.0 was considered a significant response.
Statistical analysis Analysis of DTH data was performed by means of the
Wilcoxon signed rank test or the paired difference t test. A P value of less than .05 was considered statistically significant.
RESULTS Effect of ExP on humoral immunity To assess the overall effects of ExP on humoral immunity, titers (in milligrams per deciliter) of serum immunoglobulins (IgG, IgA, and IgM) were determined before the initiation of therapy and after 6 months of therapy as described above. Six of 7 patients had total serum IgG levels within the normal range; 1 patient had elevated levels of circulating IgG. Total serum IgG levels were unchanged after 6 months of photopheresis therapy in all 7 patients (Fig 1). In addition, no changes were observed in the IgA or IgM
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Table II. Effect of extracorporeal photopheresis on cutaneous delayed-type hypersensitivity reaction to common antigens Antigen* Patient No.
1 2 3 4 5 6 7 Summary
Sample†
PPD
Trichophyton
Mumps
Candida
Tetanus
Baseline Post Baseline Post Baseline Post Baseline Post Baseline Post Baseline Post Baseline Post Median ∆‡ Paired difference§
0 0 25 8 0 0 0 0 0 0 0 0 5 0 0 P > .5
0 5 10 6 0 0 0 0 0 0 0 5 0 0 0 P > .5
0 5 0 0 5 6 8 8 0 3 10 5 0 0 0 P > .7
15 7 0 0 0 7 8 10 5 10 15 8 0 0 0 P > .8
10 8 8 0 0 0 10 0 10 0 0 2 8 5 –3.0 P > .09
*Response represented as millimeters of induration measured 48 hours postchallenge. †Baseline data obtained before initiation of photopheresis therapy; posttherapy data obtained after 6 months of photopheresis therapy. ‡Change in millimeters of induration between baseline and post-therapy measurements. §Tested by Wilcoxon signed rank test; a change of ≥ 5.0 mm from baseline was considered clinically relevant.
titers (data not shown). Furthermore, antibody titers against CMV, varicella zoster virus, and Epstein-Barr virus were not significantly changed after 6 months of therapy (Table I). Thus neither nonspecific nor specific immunoglobulin levels appeared to be altered by a 6-month course of photopheresis therapy. Antibody responses to a recall antigen, TT, were enhanced and sustained after a booster for the duration of this study. A high level of antibody response to primary antigenic challenge with KLH was observed in 3 of 3 patients tested (Fig 2). This level of response was maintained over the course of this study (minimum 6 months). Effect of ExP on cell-mediated immunity To evaluate whether ExP suppresses cell-mediated immunity, DTH skin test responses to a panel of common antigens were assessed in systemic sclerosis patients receiving this therapy. All 7 patients tested just before initiation of photopheresis displayed a positive skin test, consisting of 5 mm or more of cutaneous induration to 1 or more of the antigens (Table II). Skin test reactivity was studied after 6 months of ExP, and a change of 5 mm or more in induration from baseline was considered significant. By this criterion, 3 of the subjects who had a pretherapy response to tetanus completely lost their ability to respond to this antigen after 6 months of photopheresis. However,
there was no significant difference in the cutaneous DTH responses of the overall patient population to the test panel as determined by either the Wilcoxon signed rank test or the paired difference t test. To further determine whether ExP altered the patients’ T-cell–mediated immunity to a recall antigen, we examined the in vitro response to TT. Because administration of a booster dose of TT to patients originally enrolled in the study could interfere with the assessment of cutaneous DTH responses to TT after ExP, thus invalidating the results, 3 additional patients undergoing ExP, 2 with CTCL, received a TT booster; T-cell proliferation to the antigen was assessed in vitro at a minimum of 3 separate intervals. A significant proliferative response to TT was observed in all 3 patients tested at 1 month after booster (Table III). This response was maintained for at least 6 months in all 3 patients tested. Because it is possible that cell-mediated recall responses are less sensitive to the possible suppressive effects of ExP than are primary cell-mediated responses, we examined the patients’ ability to generate a cell-mediated immune response to KLH after primary challenge. In all 3 patients tested, a significant in vitro proliferative response was observed 1 month after challenge (Table III). This response was maintained over a period of at least 6 months in all 3 patients tested.
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Table III. In vitro T-cell proliferation responses to recall and de novo antigens in patients receiving extracorporeal photopheresis Antigen* Patient
A
B
C
Control 1
Control 2
Assay date†
Tetanus toxoid‡
KLH§
Baseline 1 mo 2 mo 3 mo 4 mo 11 mo Baseline 1 mo 8 mo 10 mo Baseline 1 mo 4 mo 6 mo 1 mo 2 mo 3 mo 1 mo 2 mo 3 mo
1.5 6.7 2.7 NT 2.0 4.5 1.0 6.3 1.0 2.2 1.0 3.7 3.0 2.0 5.3 2.4 2.8 5.8 3.3 2.9
1.0 54.1 2.1 3.3 3.4 9.7 1.0 5.3 2.2 1.0 1.0 2.7 3.3 3.2 4.2 3.3 5.8 2.8 4.7 5.0
NT, Not tested. *Response represented as stimulation index (SI) = experimental cpm/control cpm. An SI of ≥ 2.0 was considered significant. Each sample condition was assayed in triplicate. †Months after immunization. Monthly samples for assay were obtained before initiation of that month’s ExP therapy. ‡Responses to tetanus toxoid (TT) were assayed at in vitro concentrations of 10 to 50 µg/mL. Patients, but not controls received TT booster at baseline. §Patients and controls received a 5 mg immunization and subsequent 5 mg booster at 2 weeks. Responses to KLH were assayed at in vitro concentrations of 10 to 100 µg/mL.
DISCUSSION ExP has been demonstrated to be an effective therapeutic modality in the treatment of CTCL,1 for several autoimmune diseases including systemic sclerosis and pemphigus vulgaris,3,11,12 acute and chronic GVHD,5 and in the prevention of acute cellular rejection in recipients of cardiac transplants.4 Although the precise mechanism of action has not been determined, exposure of leukocytes to UVA after uptake of 8-MOP results in cross-linking of DNA and eventual apoptotic cell death.15,16 Murine skin transplantation models indicate that the treated leukocytes are altered in some fashion such that, when reinfused, a specific immunosuppressive response directed towards the treated cells occurs.10 On the basis of these observations, it would appear
that a generalized immunosuppression could occur as a result of this therapy either via induction of an immunosuppressive response or through the nonspecific loss of leukocytes. If such an effect does indeed occur, it would warrant exclusion of certain types of patients from this form of therapy to prevent exacerbation of disease via secondary infections or neoplasms. Furthermore, a generalized immunosuppression would preclude the use of various vaccines, particularly those with attenuated organisms, which are also important in maintaining the health of the patient. The present study indicates, however, that ExP does not induce a generalized immunosuppression in either the cellular or humoral component of the immune system. Our study demonstrates that patients undergoing ExP for either the treatment of scleroderma or CTCL are able to generate both Tand B-cell responses to both recall and novel antigens as evidenced by results of numerous immunologic assays. The results of this study are in contrast to those of Vonderheid et al,17 who reported that 4 of 4 patients with psoriasis who underwent photopheresis treatments lost their cutaneous DTH response and the in vitro capacity of their mononuclear cells to produce IL-2 in response to mitogens. However, the patients with psoriasis were pretreated with or received concomitant methotrexate therapy. One patient also received systemic corticosteroids along with ExP. In these patients, the generalized immunosuppression was likely because of the administration of methotrexate and corticosteroids. Quantification of serum immunoglobulin levels revealed that ExP did not suppress general or specific B-cell function. Baseline and follow-up measurements of total and specific recall Ig levels remained unchanged. When the more sensitive ELISA was used to measure secretion of antigen-specific immunoglobulins to TT and KLH, the patients universally demonstrated the ability to generate antibody responses to both the recall and novel antigens after undergoing multiple cycles of photopheresis. Results of the DTH testing showed that all patients retained cutaneous reactivity against one or more antigens. Although there appeared to be some loss of the response to specific antigens such as TT and PPD in individual patients, there was no statistically significant variation in response to any of the tested antigens among the total patient population. Nevertheless, we cannot unequivocally conclude that photopheresis does not suppress tetanus-specific responses. The ability of the patients to generate recall and primary cellular immune responses to specific antigens as assessed both in vivo (cutaneous DTH) and in vitro (T-cell proliferation assays) indi-
J AM ACAD DERMATOL VOLUME 41, NUMBER 6
cates that patients receiving ExP are not inherently more vulnerable to opportunistic or other infectious agents. Indeed, in our experience, which includes treatment of more than 125 patients in a 12-year period, there have been no incidents of opportunistic infections or neoplasms as a result of this therapy. One indicator of general immunosuppression is the reactivation of latent herpesvirus infection, which frequently occurs in the setting of the extended administration of immunosuppressive drugs. Increased antibody titers to herpesviruses suggests reactivation of latent infection and therefore poor cellular immune control of latent virus. The failure to observe a specific rise in serum antibodies against any of the herpesviruses in our study is further evidence that photopheresis does not induce a profound T-cell immunosuppression. Moreover, in a recent multicenter controlled trial using photopheresis to prevent cardiac allograft rejection, the incidence of CMV reactivation was lower in the photopheresis group than in the control group.4 Evidence that photopheresis induces specific immunity against pathogenic T-cell clones has been supported by recent observations using experimental animal models. Study of the murine model of experimental allergic encephalitis by Khavari et al18 has been especially suggestive in this regard. In this model, rats injected with myelin basic protein experience a paralytic illness associated with T-cell destruction of the nervous system. The pathogenic T cells that mediate this destruction can be isolated and cloned in vitro. When naive syngeneic rats are injected with the cloned T cells, all of the features of experimental allergic encephalitis are reproduced. However, if the pathogenic clones are first treated with psoralen and UVA and then infused, the animals are protected from the onset of disease upon subsequent challenge with the pathogenic T cells. Protection from disease appears to be mediated by the generation of clone-specific suppressor T cells that have developed in response to the psoralen and UVA-modified pathogenic cells. Similarly, Perez et al,19 using a model of cutaneous allograft rejection, have also obtained evidence of stimulation of an antigen-specific suppressor T-cell response when alloreactive effector T cells are treated with psoralen and UVA and infused into syngeneic animals.19 These results indicate that, at least in the setting of these animal models, an active immunization process can occur after administration of photoinactivated syngeneic T-cell clones and that generalized immunosuppression does not occur. Thus, the induction of anticlonotypic immunity is the presumed mechanism of action of photopheresis in the treatment of a variety of patholog-
Suchin et al 985
ic conditions including CTCL, allograft rejection, and GVHD. Although the mechanism by which ExP exerts its therapeutic effects is not wholly appreciated, these data demonstrate that its efficacy is not a result of generalized immunosuppression. This may explain the observation that patients undergoing ExP have no greater risk of experiencing opportunistic infections or neoplasms compared with the normal population. In addition, it suggests that attenuated live vaccines may be used in the preventive health management of patients receiving photopheresis. Based on our results we recommend administration of vaccines at the conclusion of a 2-day treatment cycle. REFERENCES 1. Edelson R, Berger C, Gasparro F, Jegasothy B, Heald P, Wintroub B, et al. Treatment of cutaneous T-cell lymphoma by extracorporeal photochemotherapy: preliminary results. N Engl J Med 1987;316:297-303. 2. Gottleib SL, Wolfe JT, Fox FE, DeNardo BJ, Macey WH, Bromley PG, et al. Treatment of cutaneous T-cell lymphoma with extracorporeal photopheresis monotherapy and in combination with recombinant interferon alpha: a 10-year experience at a single institution. J Am Acad Dermatol 1996;35:946-57. 3. Rook AH, Freundlich B, Jegasothy BV, Perez MI, Barr WG, Jimenez SA, et al. Treatment of systemic sclerosis with extracorporeal photochemotherapy: results of a multicenter trial. Arch Dermatol 1992;128:337-46. 4. Barr ML, Meiser BM, Eisen HJ, Roberts RF, Livi U, Dall’Amico R, et al. Photopheresis for the prevention of rejection in cardiac transplantation. N Engl J Med 1998;339:1744-50. 5. Greinix HT, Volc-Platzer B, Rabitsch W, Gmeinhart B, GuevaraPineda C, Kalhs P, et al. Successful use of extracorporeal photochemotherapy in the treatment of severe acute and chronic graft-versus-host disease. Blood 1998;92:3098-104. 6. Vowels BR, Cassin M, Boufal MH, Walsh LJ, Rook AH. Extracorporeal photochemotherapy induces the production of tumor necrosis factor-alpha by monocytes: implications for the treatment of cutaneous T-cell lymphoma and systemic sclerosis. J Invest Dermatol 1992;98:686-92. 7. Kraemer KH, Waters HL, Cohen LF, Popescu NC, Amsbaugh SC, DiPaolo JA, et al. Effects of 8-methoxypsoralen and ultraviolet radiation on human lymphoid cells in vitro. J Invest Dermatol 1981;76:80-7. 8. Berger CL, Cantor C, Welsh J, Dervan P, Begley T, Grant S, et al. Comparison of synthetic psoralen derivatives and 8-MOP in the inhibition of lymphocyte proliferation. Ann NY Acad Sci 1985; 453:80-90. 9. Morison WL, Wimberly J, Parrish JA, Bloch KJ. Abnormal lymphocyte function following long-term PUVA therapy for psoriasis. Br J Dermatol 1983;108:445-50. 10. Perez M, Edelson R, Laroche L, Berger C. Inhibition of antiskin allograft immunity by infusions with syngeneic photoinactivated effector lymphocytes. J Invest Dermatol 1989;92:669-76. 11. Rook AH, Freundlich B, Nahass GT,Washko R, Macelis B, Skolnicki M, et al. Treatment of autoimmune disease with extracorporeal photochemotherapy: progressive systemic sclerosis. Yale J Biol Med 1989;62:639-45. 12. Rook AH. Photopheresis in the treatment of autoimmune disease: experience with pemphigus vulgaris and systemic sclerosis. Ann NY Acad Sci 1991;636:209-16. 13. Ullrich SE. Photoinactivation of T-cell function with psoralen
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and UVA radiation suppresses the induction of experimental murine graft-versus-host disease across major histocompatibility barriers. J Invest Dermatol 1991;96:303-8. 14. Stallone DD, Stunkard AJ, Zweiman B, Wadden TA, Foster GD. Decline in delayed type hypersensitivity response in obese women following weight reduction. Clin Diagnostic Lab Immunol 1994;1:202-5. 15. Gasparro FP, Dall’Amico R, Goldminz D, Simmons E, Weingold D. Molecular aspects of extracorporeal photochemotherapy. Yale J Biol Med 1989;62:579-93. 16. Yoo EK, Rook AH, Elenitsas R, Gasparro FP, Vowels BR. Apoptosis induction by ultraviolet light A and photochemotherapy in
O
cutaneous T-cell lymphoma: relevance to mechanism of therapeutic action. J Invest Dermatol 1996;107:235-42. 17. Vonderheid EC, Bigler RD, Rogers TJ, Kadin ME, Griffin TD. Effect of extracorporeal photopheresis on selected immunologic parameters in psoriasis vulgaris.Yale J Biol Med 1989;62:653-64. 18. Khavari PA, Edelson RL, Lider O, Gasparro FP, Weiner HL, Cohen IR. Specific vaccination against photoinactivated cloned T-cells. Clin Res 1988;36:662. 19. Perez M, Edelson R, LaRoche L, Berger C. Specific suppression of antiallograft immunity by immunization with syngeneic photoinactivated effector lymphocytes. J Invest Dermatol 1989;92: 669-76.
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