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CD4+CD7− T cells compose the dominant T-cell clone in the peripheral blood of patients with Sézary syndrome
CD4+CD7– T cells compose the dominant T-cell clone in the peripheral blood of patients with Sézary syndrome Gunter Rappl, PhD,a Joachim Marcus Muche, MD,b Hinrich Abken, MD, PhD,c Wolfram Sterry, MD,b Wolfgang Tilgen, MD,a Selma Ugurel, MD,a and Uwe Reinhold, MDa Homburg/Saar, Berlin, and Cologne, Germany Background: Absence of CD7 antigen expression in T cells defines a subset of normal CD4+ CD45RO+ CD45RA– memory cells and is furthermore observed in Sézary syndrome (SS). Objective: Our purpose was to identify circulating T-cell clones in patients with SS and to elucidate whether the dominant T-cell clones express the CD7 antigen. Methods: Peripheral blood lymphocytes of patients with SS were analyzed by two-color flow cytometry using antibodies to the Vβ region of the T cell receptor (TCR) in combination with an antibody to CD7. In addition, T cells were analyzed for TCR-γ gene rearrangement by polymerase chain reaction (PCR) techniques. Results: Clonal T-cell expansion was detected in 7 patients with SS by immunostaining of the TCR Vβ regions. PCR analysis confirmed the presence of dominant T cell clones. Double-immunostaining revealed that in each case cells of the clonal Vβ TCR rearrangement homogeneously express the CD4+CD7– phenotype. Furthermore, CD4+CD7– cells express the CD15s antigen but lack expression of CD26 and CD49d. Conclusion: Expansion of clonal T cells strongly correlates with the expansion of CD4+CD7– T cells in 7 tested patients with SS. This supports our model that a subset of late differentiated, normal CD4+CD7– memory T cells may represent the physiologic counterpart of Sézary cells. Monitoring of circulating T cells with the CD4+CD7–CD15s+CD26–CD49d– phenotype proved to be useful for the identification of clonal T cells in patients with SS. (J Am Acad Dermatol 2001;44:456-61.)
T
he prototype of cutaneous T-cell lymphoma (CTCL) is the cerebriform T-cell lymphoma, which is historically subdivided into mycosis fungoides (MF) and Sézary syndrome (SS). MF typically presents as cutaneous patches that can progress to infiltrated plaques and ultimately cutaneous tumors with lymphoid and visceral involvement. In SS, skin
From the Department of Dermatology, The Saarland University Hospital, Homburg/Saar,a the Department of Dermatology and Allergy,b University Hospital Charité, Humboldt University Berlin,b and the Laboratory for Tumorgenetics and Cell Biology, Department of Internal Medicine, University of Cologne.c Supported by the Deutsche Forschungsgemeinschaft DFG (to U. R.; Re 690/4-2) and the Fritz-Bender Stiftung, Munich (to H. A.). Accepted for publication Aug 15, 2000. Reprint requests: Uwe Reinhold, MD, Department of Dermatology, The Saarland University Hospital, 66421 Homburg/Saar, Germany. Copyright © 2001 by the American Academy of Dermatology, Inc. 0190-9622/2001/$35.00 + 0 16/1/110900 doi:10.1067/mjd.2001.110900
456
Abbreviations used: CTCL: mAb: MF: PBMC: PCR: SS: TCR:
cutaneous T-cell lymphoma monoclonal antibody mycosis fungoides peripheral blood mononuclear cells polymerase chain reaction Sézary syndrome T-cell receptor
(erythroderma), blood, lymph nodes, spleen, and liver are progressively involved. The most characteristic feature of SS is the presence of leukemic T cells (Sézary cells) with a unique micromorphology, that is, a nucleus with a cerebriform contour and a high nucleus-tocytoplasm ratio.1,2 Most intriguing, the immunophenotype of Sézary cells is frequently indistinguishable from mature CD4+CD45RO+CD45RA– “memory” T cells.3 Occasional cases, however, are reported to be of
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CD4–CD8+ or CD4+CD8+ phenotype.1 Of particular interest is the absence of CD7 antigen expression on a high percentage of leukemic and skin-infiltrating T cells in patients with cerebriform T-cell lymphoma.4,5 It has been demonstrated that the number of circulating CD4+CD7– T cells in SS strongly correlates with the number of morphologically identified Sézary cells.5 A recent report furthermore showed that the absence of CD7 antigen expression on CD4+ T cells is the only known immunohistochemical criterion that discriminates between MF and control cases.6 Because phenotype aberrations by loss of surface antigens are common findings in malignant lymphoma, the lack of CD7 antigen expression on CTCL cells was thought to be caused by a further transformation of the neoplastic cells.5 Over the last years, however, it became increasingly evident that absence of CD7 antigen defines a particular subset of normal late-differentiated CD4+ memory T cells that expand during certain physiologic and pathologic conditions in vivo.3,7,8 The accumulation of CD4+CD7– cells in nonlymphatic tissues and the expression of homing receptors such as the cutaneous lymphocyte antigen imply a preferential organ-specific homing of the CD7– memory T-cell subset.9 Because the majority of normal dermal and epidermal T cells and locally accumulated CD4+ T cells in a variety of benign inflammatory skin lesions are of the CD7– phenotype, the homing of this T-cell subset seems to be directed to the cutaneous compartment.7,10,11 Absence of CD7 antigen expression in normal or malignant CD4+ T cells reflects a stable differentiation status because repeated in vitro stimulation of naive CD4+ T cells leads to the development of the CD7– memory subset that does not revert to the CD7+ phenotype even after prolonged propagation.11 The data presently available strongly suggest that normal CD4+CD7– cells may represent the physiologic counterpart of CTCL cells, at least in certain cases.3 Whereas expansion of CD4+CD7– has been observed in patients with benign inflammatory skin disease,7 it remains to be determined whether CD4+CD7– T cells represent the neoplastically transformed cell population in SS. Using a panel of antibodies to the variable regions of the T cell receptor (TCR) β chain, we were able to test whether the CD4+CD7– subset in the peripheral blood of 7 patients with SS accurately identifies the clonally expanded T-cell population.
patients with SS were recruited for the study. After informed consent was obtained, blood was obtained via venipuncture, and portions of each sample were used for the following purposes: a complete blood cell count, a Sézary cell count as determined by light microscopy, isolation of peripheral blood mononuclear cells (PBMCs) by standard Ficoll-Hypaque density centrifugation, and genomic DNA extraction. Immunophenotyping Flow cytometry analyses were performed by single-color and two-color immunofluorescence using fluorescein isothiocyanate and phycoerythrinconjugated monoclonal antibodies (mAbs), respectively. Staining with irrelevant isotype-matched antibodies were used as control. The following antibodies were used: Leu-3a (CD4), Leu-2a (CD8) (Becton Dickinson, Heidelberg, Germany), 3A1 (CD7) (Sigma, Deisenhofen, Germany), CD15s (sialyl LewisX), CD26, CD49d (Immunotech, Hamburg, Germany). A panel of 16 mAbs to human TCR Vβ regions (Vβ2, Vβ3, Vβ5.1, Vβ5.2, Vβ6.1, Vβ8, Vβ11, Vβ12, Vβ13.1, Vβ13.6, Vβ14, Vβ16, Vβ17, Vβ20, Vβ21.3, Vβ22) was obtained from Immunotech. TCR γ gene rearrangement studies Genomic DNA was prepared from l × 106 PBMC by standard procedures using “High Pure Template Preparation Kit” (Roche Diagnostics, Mannheim, Germany). Rearranged TCR γ gene fragments were amplified by means of specific consensus primer oligonucleotides for the variable (V) and joining (J) regions as previously reported.12 Primer oligonucleotides for amplification of the β-globin DNA are described elsewhere.13 The reaction mixture was composed of 0.5 to 1 µg (5 µL) genomic DNA, 1.75 U Taq polymerase, 10× polymerase chain reaction (PCR) buffer (7.5 µL; Perkin Elmer, Weiterstadt, Germany), 0.1 mmol/L of each deoxynucleotide triphosphate (dNTP; Gibco, Karlsruhe, Germany), and 0.6 µmol/L of each primer in a final volume of 75 µL. Amplification was performed on a thermal cycler (2400 Gene Amp, Perkin Elmer) by a 4-minute denaturation step at 95°C, followed by 40 cycles of 1 minute at 94°C, 1 minute at 54°C, and 1 minute at 72°C. A final extension step was performed for 7 minutes at 72°C. Reaction products were analyzed by polyacrylamide gel electrophoresis (8%) according to standard protocols.
RESULTS MATERIAL AND METHODS Patients The diagnosis of SS was established by characteristic erythroderma, diagnostic skin biopsy specimen, and blood Sézary cell count of more than 1000/µL. Seven
Detection of clonal T cells by anti-TCR Vβ monoclonal antibodies Blood samples obtained from 7 patients with SS were immunophenotypically analyzed by flow cytometry. All patients had a significantly increased per-
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Table I. Immunophenotyping of peripheral blood T cells in patients with SS Immunophenotype Patient No.
Sex/ Age (y)
1 2 3 4 5 6 7 Normal range
M/75 M/64 M/70 F/69 M/48 F/65 F/58
CD3 %(cells/µL)
CD4 %(cells/µL)
CD8 %(cells/µL)
98 (4686) 82 (3920) 16 (755) 99 (11,724) 98 (11,357) 1 (244) 99 (10,820) 97 (10,432) 2 (267) 87 (4271) 84 (3900) 3 (289) 78 (8375) 74 (7913) 4 (301) 91 (15,197) 91 (15,197) 0 98 (7487) 96 (7334) 2 (152) 65%-81% 35%-56% 17%-36% (950-2000/µL) (550-1200/µL) (500-1050/µL)
CD4/CD8 ratio
CD4+CD7– %
CD4+CD7+ %
5.1 98.0 48.5 28.0 18.5 — 38.5 1.1-2.9
77 (3681) 5 (239) 96 (11,126) 2 (231) 92 (9895) 5 (537) 81 (3761) 3 (139) 72 (7699) 2 (214) 91 (15,197) 0 77 (5882) 19 (1452) < 10% 35%-56% (< 150 µL) (550-1200 µL)
Sézary cell count (%)
52 70 65 39 20 66 50 < 10%
Table II. Detection of the dominant T-cell clone by immunofluorescence analysis Two-color immunofluorescence (% positive cells) Patient No.
Vβ clone (% of CD3+ cells)
Vβ+CD7–
Vβ+CD7+
Vβ+CD15s+
Vβ+CD26–
Vβ+CD49d–
1 2 3 4 5 6 7
Vβ12 (77) Vβ13.1 (96) Vβ11 (92) Vβ3 (81) Vβ20 (72) Vβ20 (91) Vβ17 (77)
> 98 > 98 > 98 > 98 > 98 > 98 > 98
<2 <2 <2 <2 <2 <2 <2
NT 96 96 96 98 96 99
NT 96 97 96 97 96 98
NT 98 98 98 97 97 99
NT, Not tested.
centage (P < .001) and an increased absolute number of CD4+ T cells compared with standard control values (Table I). Accordingly, all patients had an abnormal increase in the CD4/CD8 ratio (mean, 46.8). The TCR Vβ repertoire was analyzed by means of a panel of Vβ specific antibodies. As summarized in Table II, a dominant and markedly expanded T-cell population, specified by the particular Vβ usage and constituting 77% to 96% of total T cells, was found in blood samples of each patient tested. It is noteworthy that the Sézary cell count underestimated the number of clonal cells in all cases studied. To confirm whether the expanded Vβ T cell population constitutes a homogeneous T-cell clone, we analyzed the TCR γ chain rearrangement by PCR. A clonal TCR γ chain rearrangement was found in blood samples of all 7 patients (Fig 1). Taken together, we conclude that the dominant Vβ T cells in the peripheral blood of the SS patients are of clonal origin. Double-immunofluorescence analysis We furthermore asked whether the dominant Tcell clones in the peripheral blood of patients with SS express the CD7 antigen. Double-immunofluores-
cence staining of PBMC revealed that the percentage of circulating CD4+CD7– T cells is significantly increased compared with healthy controls (Table I). CD8+CD7– T cells were not detectable in blood samples of any of the patients (data not shown). By twocolor immunofluorescence, we analyzed whether cells of the dominant Vβ T-cell clone express the CD7 antigen. As shown in a representative analysis in Fig 2, almost all cells of the dominant Vβ clone do not express the CD7 antigen. This observation is consistent for all 7 patients tested (Table II) demonstrating that the number of clonal T cells in the peripheral blood of these patients with SS precisely matches the number of CD4+CD7– T cells. We next analyzed the coexpression of CD15s, CD26, and CD49d antigens on clonal Vβ+ T cells. As shown in Table II, the clonal Vβ+CD7– cells homogeneously express the CD15s antigen whereas the percentage of Vβ+ T cells expressing CD26 or CD49d antigen, respectively, was less than 5% in all patients tested.
DISCUSSION Several reports showed that the CD4+CD7– cell population is expanded in the blood of patients with
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Fig 1. DNA from peripheral blood lymphocytes was subjected to PCR amplification of the rearranged TCR γ chain. PCR products were analyzed by polyacrylamide gel electrophoresis. Lane 1, Size marker of DNA; lane 2, negative control (normal blood lymphocytes); lane 3, positive control (Jurkat cells); lanes 4-10, blood samples of patients with SS.
Fig 2. Representative double-immunofluorescence staining of blood lymphocytes from a patient with SS (patient 4). Peripheral blood lymphocytes were stained with anti-Vβ3 and with anti-CD7 monoclonal antibodies and analyzed by flow cytometry. Note expansion of population of Vβ3+ CD7– T cells.
SS and suggested, although not formally proved, that these cells represent the malignant cell clone.4,5 This assumption, however, has been recently called into question by the observation of clonal T cells in both the CD7+ and CD7– population of leukemic Sézary cells.14 The present study was undertaken to evaluate whether the CD4+CD7– immunophenotype is associated with cells of the dominant T-cell clone in the peripheral blood of patients with SS. We identified the dominant T-cell clone in 7 patients with SS by means of a panel of mAbs directed to var-
ious TCR Vβ regions. The detection of clonal TCR γ gene rearrangements by PCR confirmed the presence of a T-cell clone in all 7 cases. Double-immunofluorescence studies revealed that nearly all (98%) clonal TCR Vβ T cells in all 7 patients tested express the CD4+CD7– phenotype suggesting that the dominant T-cell clone originates from the CD4+CD7– Tcell subset. The results are in agreement with the finding that the CD4+CD7– T-cell subset expands in the majority of patients with CTCL.4,5,15 The data are also compatible with the observation that the num-
460 Rappl et al
ber of CD4+CD7– T cells and the number of clonal T cells identified by anti-Vβ6.7 mAbs were found identical in a patient with SS over a period of 6 months.4 Our results confirm a previous study showing that expansion of circulating CD4+CD7– cells in advanced stages of cutaneous T-cell lymphoma is associated with CD15s expression and lack of CD26 and CD49d expression.15 It remains to be determined whether the CD15s+CD26–CD49d– immunophenotype corresponds to a discrete subpopulation of mature CD4+CD7– T cells. In this context, strong expression of sialyl LewisX (CD15s) antigens on peripheral T cells has been recently observed in kidney transplant recipients with rejection indicating that CD15s expression on T cells correlates with persistent antigen stimulation in vivo.16 The following observations support the concept that the CD4+CD7– T-cell subset may represent a candidate counterpart of certain forms of CTCL: (1) The vast majority of leukemic T cells in the peripheral blood of patients with SS lack CD7 antigen expression but show high expression of cutaneous lymphocyte antigen4,5,17; (2) cutaneous infiltrates of lymphoid cells in SS and other types of CTCL contain a high number of CD4+CD7– T cells6; (3) the surface antigen CH-F42 is expressed by normal CD4+CD7– T cells and malignant CD4+CD7– Sézary cells but is absent in cells of a variety of other hematologic malignancies18; (4) physiologic CD4+CD7– T cells show a TH0/TH2-like cytokine secretion pattern and a preferential production of interleukin 5 as do malignant T cells of CTCL with TH2 activities.19-21 We therefore suggest that the very similar properties of physiologic and neoplastic CD4+CD7– T cells make the assumption unlikely that loss of CD7 expression in CTCL is the result of an unspecific antigen loss (“aberrant” phenotype) during malignant transformation.6 Our results are in apparent discrepancy to a study reported by Dummer et al14 and their general conclusion that the CD4+CD7– population does not represent the dominant T-cell clone in patients with SS. Whereas their conclusion is based on a very low number of patients, we tested circulating blood cells of 7 patients with SS by means of double-immunofluorescence analysis and consistently obtained adverse results. Our data, however, are in agreement with their recent results showing that the CD4+ CD7– immunophenotype correlates with the detection of clonal cells in the peripheral blood by PCR.22 Although a minority of patients with CD7+ SS have been described,23 absence of CD7 antigen is generally accepted to be a common feature of malignant Sézary cells. The reasons for the discrepant observations remain unresolved so far. However, it will be worthwhile to ask whether there are immunologic
J AM ACAD DERMATOL MARCH 2001
or clinical differences between the majority of SS with a CD7– phenotype and those minor cases with CD7 antigen expression. In conclusion, the present study shows that the CD4+CD7– immunophenotype is strongly associated with the dominant T-cell clone in the peripheral blood of 7 analyzed patients with SS. The results confirm and extend previous reports indicating that the flow cytometric enumeration of CD4+CD7– T cells and the analysis of expression of CD15s versus nonexpression of CD26 and CD49d antigens provides useful information for the diagnosis and follow-up in patients with SS.24-26 The results further support the concept that the majority of Sézary cells correspond to a physiologic CD4+CD7– counterpart of mature T cells. We thank S. Maas and K. Hilgert for great technical assistance, and Dr R. Gold (Würzburg) for supplying us with TCR Vβ– monoclonal antibodies. REFERENCES 1. Diamandidou E, Cohen PR, Kurzrock R. Mycosis fungoides and Sézary syndrome. Blood 1996;88:2385-409. 2. Willemze R, Sterry W, Berti E, Cerroni L, Chimenti S, Diaz-Pérez JL, et al. EORTC classification for primary cutaneous lymphoma: a proposal from the cutaneous study group of the European organisation for research and treatment of cancer. Blood 1997; 90:354-71. 3. Reinhold U, Abken H. CD4+ CD7– T cells: a separate subpopulation of memory T cells? J Clin Immunol 1997;17:265-71. 4. Bogen SA, Pelley D, Charif M, McCusher M, Koh H, Foss F, et al. Immunophenotypic identification of Sézary cells in peripheral blood. Am J Clin Pathol 1996;106:739-48. 5. Harmon CB, Witzig TE, Katzmann JA, Pittelkow MR. Detection of circulating T cells with CD4+ CD7– immunophenotype in patients with benign and malignant lymphoproliferative dermatoses. J Am Acad Dermatol 1996;35:404-10. 6. Bergman R, Faclieru D, Sahar D, Sander CA, Kerner H, Ben-Aryeh Y, et al. Immunophenotyping and T cell receptor γ gene rearrangement analysis as an adjunct to the histopathologic diagnosis of mycosis fungoides. J Am Acad Dermatol 1998;39: 554-9. 7. Moll M, Reinhold U, Kukel S, Abken H, Müller R, Oltermann I, et al. CD7 negative helper T cells accumulate in inflammatory skin lesions. J Invest Dermatol 1994;102:328-32. 8. Reinhold U, Abken H, Kukel S, Moll M, Oltermann I, Müller R, et al. CD7– T cells represent a subset of normal human blood lymphocytes. J Immunol 1993;150:2081-9. 9. Reinhold U, Liu L, Sesterhenn J, Abken H. CD7-negative T cells represent a separate differentiation pathway in a subset of post-thymic helper T cells. Immunology 1996;89:391-6. 10. Foster CA,Yokozeki H, Rappersberger K, Koning F,Volc-Platzer B, Rieger A, et al. Human epidermal T cells predominantly belong to the lineage expressing alpha/beta T cell receptor. J Exp Med 1990;171:997-1013. 11. Davis AL, McKenzie JL, Hart DN. HLA-DR–positive leucocyte subpopulations in human skin include dendritic cells, macrophages, and CD7-negative T cells. Immunology 1988;65: 573-81. 12. Muche JM, Lukowsky A, Asadullah K, Gellrich S, Sterry W. Demonstration of frequent occurrence of clonal T cells in the
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13.
14.
15.
16.
17.
18.
19.
peripheral blood of patients with primary cutaneous lymphoma. Blood 1997;90:1636-42. Serfling M, Lukowsy A, Audring H, Sterry W. Search for Kaposi’s sarcoma–associated virus DNA in hemangioproliferative disorders and cutaneous malignant lymphoma. J Cutan Pathol 1997; 24:298-304. Dummer R, Nestle FO, Niederer E, Ludwig E, Laine E, Grundmann H, et al. Genotypic, phenotypic and functional analysis of CD4+ CD7+ and CD4+ CD7– T lymphocyte subsets in Sézary syndrome. Arch Dermatol Res 1999;291:307-11. Scala E, Russo G, Cadoni S, Narducci MG, Girardelli CR, De Pità O, et al. Skewed expression of activation, differentiation and homing-related antigens in circulating cells from patients with cutaneous T cell lymphoma associated with CD7– T helper lymphocyte expansions. J Invest Dermatol 1999;113:622-7. Ishida H, Koyama I, Sawada T, Murakami T, Utsumi K, Sannomiya A, et al. Clinical utility of monitoring sialyl Lewis (X) (CD15s) antigen on peripheral lymphocytes for the diagnosis and treatment of rejection after renal transplantation. Transplantation 2000;69:59-63. Heald PW,Yan SL, Edelson RL,Tigelaar R, Picker LJ. Skin-selective lymphocyte homing mechanisms in the pathogenesis of leukemic cutaneous T-cell lymphoma. J Invest Dermatol 1993; 101:222-6. Labastide WB, Rana MT, Barker CR. A new monoclonal antibody (CH-F42) recognizes a CD7– subset of normal T lymphocytes and circulating malignant cells in adult T-cell lymphoma– leukemia and Sézary syndrome. Blood 1990;76:1361-8. Reinhold U, Liu L, Sesterhenn J, Schnautz S, Abken H. The CD7– subset represents the majority of IL-5 secreting cells within CD4+ CD45RA– T cells. Clin Exp Immunol 1996;106:555-9.
20. Autran B, Legac E, Blanc C, Debré P. A Th0/Th2-1ike function of CD4+ CD7– T helper cells from normal donors and HIV-infected patients. J Immunol 1995;154:1408-17. 21. Dummer R, Heald PW, Nestle FO, Ludwig E, Laine E, Hemmi S, et al. Sézary syndrome’s T cell clones display T helper 2 cytokines and express the accessory factor-1 (interferon gamma receptor beta chain). Blood 1996;88:1383-9. 22. Laetsch B, Haffner AC, Dobbeling U, Seifert B, Ludwig E, Burg G, et al. CD4+/CD7– T cell frequency and polymerase chain reaction-based clonality assay correlate with stage in cutaneous T cell lymphomas. J Invest Dermatol 2000;114:107-11. 23. Yagi H, Tokura Y, Furukawa F, Takigawa M. CD7-positive Sézary syndrome with a Th1 cytokine profile. J Am Acad Dermatol 1996;34:368-74. 24. Fritz TM, Kleinhans M, Nestle FO, Burg G, Dummer R. Combination treatment with extracorporeal photopheresis, interferon alfa and interleukin-2 in a patient with the Sézary syndrome. Br J Dermatol 1999;140:1144-7. 25. Bernengo MG, Quaglino P, Novelli M, Cappello N, Doveil GC, Lisa F, et al. Prognostic factors in Sézary syndrome: a multivariate analysis of clinical, haematological and immunological features. Ann Oncol 1998;9:857-63. 26. Collenberg C, von den Driesch P, Gramatzki M, Fartasch M, Gruschwitz MS. Monitoring of the disease progress in Sézary syndrome by CD7– cells using flow cytometry. Br J Dermatol 1996;134:980-2.
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T
he prototype of cutaneous T-cell lymphoma (CTCL) is the cerebriform T-cell lymphoma, which is historically subdivided into mycosis fungoides (MF) and Sézary syndrome (SS). MF typically presents as cutaneous patches that can progress to infiltrated plaques and ultimately cutaneous tumors with lymphoid and visceral involvement. In SS, skin
From the Department of Dermatology, The Saarland University Hospital, Homburg/Saar,a the Department of Dermatology and Allergy,b University Hospital Charité, Humboldt University Berlin,b and the Laboratory for Tumorgenetics and Cell Biology, Department of Internal Medicine, University of Cologne.c Supported by the Deutsche Forschungsgemeinschaft DFG (to U. R.; Re 690/4-2) and the Fritz-Bender Stiftung, Munich (to H. A.). Accepted for publication Aug 15, 2000. Reprint requests: Uwe Reinhold, MD, Department of Dermatology, The Saarland University Hospital, 66421 Homburg/Saar, Germany. Copyright © 2001 by the American Academy of Dermatology, Inc. 0190-9622/2001/$35.00 + 0 16/1/110900 doi:10.1067/mjd.2001.110900
456
Abbreviations used: CTCL: mAb: MF: PBMC: PCR: SS: TCR:
cutaneous T-cell lymphoma monoclonal antibody mycosis fungoides peripheral blood mononuclear cells polymerase chain reaction Sézary syndrome T-cell receptor
(erythroderma), blood, lymph nodes, spleen, and liver are progressively involved. The most characteristic feature of SS is the presence of leukemic T cells (Sézary cells) with a unique micromorphology, that is, a nucleus with a cerebriform contour and a high nucleus-tocytoplasm ratio.1,2 Most intriguing, the immunophenotype of Sézary cells is frequently indistinguishable from mature CD4+CD45RO+CD45RA– “memory” T cells.3 Occasional cases, however, are reported to be of
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CD4–CD8+ or CD4+CD8+ phenotype.1 Of particular interest is the absence of CD7 antigen expression on a high percentage of leukemic and skin-infiltrating T cells in patients with cerebriform T-cell lymphoma.4,5 It has been demonstrated that the number of circulating CD4+CD7– T cells in SS strongly correlates with the number of morphologically identified Sézary cells.5 A recent report furthermore showed that the absence of CD7 antigen expression on CD4+ T cells is the only known immunohistochemical criterion that discriminates between MF and control cases.6 Because phenotype aberrations by loss of surface antigens are common findings in malignant lymphoma, the lack of CD7 antigen expression on CTCL cells was thought to be caused by a further transformation of the neoplastic cells.5 Over the last years, however, it became increasingly evident that absence of CD7 antigen defines a particular subset of normal late-differentiated CD4+ memory T cells that expand during certain physiologic and pathologic conditions in vivo.3,7,8 The accumulation of CD4+CD7– cells in nonlymphatic tissues and the expression of homing receptors such as the cutaneous lymphocyte antigen imply a preferential organ-specific homing of the CD7– memory T-cell subset.9 Because the majority of normal dermal and epidermal T cells and locally accumulated CD4+ T cells in a variety of benign inflammatory skin lesions are of the CD7– phenotype, the homing of this T-cell subset seems to be directed to the cutaneous compartment.7,10,11 Absence of CD7 antigen expression in normal or malignant CD4+ T cells reflects a stable differentiation status because repeated in vitro stimulation of naive CD4+ T cells leads to the development of the CD7– memory subset that does not revert to the CD7+ phenotype even after prolonged propagation.11 The data presently available strongly suggest that normal CD4+CD7– cells may represent the physiologic counterpart of CTCL cells, at least in certain cases.3 Whereas expansion of CD4+CD7– has been observed in patients with benign inflammatory skin disease,7 it remains to be determined whether CD4+CD7– T cells represent the neoplastically transformed cell population in SS. Using a panel of antibodies to the variable regions of the T cell receptor (TCR) β chain, we were able to test whether the CD4+CD7– subset in the peripheral blood of 7 patients with SS accurately identifies the clonally expanded T-cell population.
patients with SS were recruited for the study. After informed consent was obtained, blood was obtained via venipuncture, and portions of each sample were used for the following purposes: a complete blood cell count, a Sézary cell count as determined by light microscopy, isolation of peripheral blood mononuclear cells (PBMCs) by standard Ficoll-Hypaque density centrifugation, and genomic DNA extraction. Immunophenotyping Flow cytometry analyses were performed by single-color and two-color immunofluorescence using fluorescein isothiocyanate and phycoerythrinconjugated monoclonal antibodies (mAbs), respectively. Staining with irrelevant isotype-matched antibodies were used as control. The following antibodies were used: Leu-3a (CD4), Leu-2a (CD8) (Becton Dickinson, Heidelberg, Germany), 3A1 (CD7) (Sigma, Deisenhofen, Germany), CD15s (sialyl LewisX), CD26, CD49d (Immunotech, Hamburg, Germany). A panel of 16 mAbs to human TCR Vβ regions (Vβ2, Vβ3, Vβ5.1, Vβ5.2, Vβ6.1, Vβ8, Vβ11, Vβ12, Vβ13.1, Vβ13.6, Vβ14, Vβ16, Vβ17, Vβ20, Vβ21.3, Vβ22) was obtained from Immunotech. TCR γ gene rearrangement studies Genomic DNA was prepared from l × 106 PBMC by standard procedures using “High Pure Template Preparation Kit” (Roche Diagnostics, Mannheim, Germany). Rearranged TCR γ gene fragments were amplified by means of specific consensus primer oligonucleotides for the variable (V) and joining (J) regions as previously reported.12 Primer oligonucleotides for amplification of the β-globin DNA are described elsewhere.13 The reaction mixture was composed of 0.5 to 1 µg (5 µL) genomic DNA, 1.75 U Taq polymerase, 10× polymerase chain reaction (PCR) buffer (7.5 µL; Perkin Elmer, Weiterstadt, Germany), 0.1 mmol/L of each deoxynucleotide triphosphate (dNTP; Gibco, Karlsruhe, Germany), and 0.6 µmol/L of each primer in a final volume of 75 µL. Amplification was performed on a thermal cycler (2400 Gene Amp, Perkin Elmer) by a 4-minute denaturation step at 95°C, followed by 40 cycles of 1 minute at 94°C, 1 minute at 54°C, and 1 minute at 72°C. A final extension step was performed for 7 minutes at 72°C. Reaction products were analyzed by polyacrylamide gel electrophoresis (8%) according to standard protocols.
RESULTS MATERIAL AND METHODS Patients The diagnosis of SS was established by characteristic erythroderma, diagnostic skin biopsy specimen, and blood Sézary cell count of more than 1000/µL. Seven
Detection of clonal T cells by anti-TCR Vβ monoclonal antibodies Blood samples obtained from 7 patients with SS were immunophenotypically analyzed by flow cytometry. All patients had a significantly increased per-
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Table I. Immunophenotyping of peripheral blood T cells in patients with SS Immunophenotype Patient No.
Sex/ Age (y)
1 2 3 4 5 6 7 Normal range
M/75 M/64 M/70 F/69 M/48 F/65 F/58
CD3 %(cells/µL)
CD4 %(cells/µL)
CD8 %(cells/µL)
98 (4686) 82 (3920) 16 (755) 99 (11,724) 98 (11,357) 1 (244) 99 (10,820) 97 (10,432) 2 (267) 87 (4271) 84 (3900) 3 (289) 78 (8375) 74 (7913) 4 (301) 91 (15,197) 91 (15,197) 0 98 (7487) 96 (7334) 2 (152) 65%-81% 35%-56% 17%-36% (950-2000/µL) (550-1200/µL) (500-1050/µL)
CD4/CD8 ratio
CD4+CD7– %
CD4+CD7+ %
5.1 98.0 48.5 28.0 18.5 — 38.5 1.1-2.9
77 (3681) 5 (239) 96 (11,126) 2 (231) 92 (9895) 5 (537) 81 (3761) 3 (139) 72 (7699) 2 (214) 91 (15,197) 0 77 (5882) 19 (1452) < 10% 35%-56% (< 150 µL) (550-1200 µL)
Sézary cell count (%)
52 70 65 39 20 66 50 < 10%
Table II. Detection of the dominant T-cell clone by immunofluorescence analysis Two-color immunofluorescence (% positive cells) Patient No.
Vβ clone (% of CD3+ cells)
Vβ+CD7–
Vβ+CD7+
Vβ+CD15s+
Vβ+CD26–
Vβ+CD49d–
1 2 3 4 5 6 7
Vβ12 (77) Vβ13.1 (96) Vβ11 (92) Vβ3 (81) Vβ20 (72) Vβ20 (91) Vβ17 (77)
> 98 > 98 > 98 > 98 > 98 > 98 > 98
<2 <2 <2 <2 <2 <2 <2
NT 96 96 96 98 96 99
NT 96 97 96 97 96 98
NT 98 98 98 97 97 99
NT, Not tested.
centage (P < .001) and an increased absolute number of CD4+ T cells compared with standard control values (Table I). Accordingly, all patients had an abnormal increase in the CD4/CD8 ratio (mean, 46.8). The TCR Vβ repertoire was analyzed by means of a panel of Vβ specific antibodies. As summarized in Table II, a dominant and markedly expanded T-cell population, specified by the particular Vβ usage and constituting 77% to 96% of total T cells, was found in blood samples of each patient tested. It is noteworthy that the Sézary cell count underestimated the number of clonal cells in all cases studied. To confirm whether the expanded Vβ T cell population constitutes a homogeneous T-cell clone, we analyzed the TCR γ chain rearrangement by PCR. A clonal TCR γ chain rearrangement was found in blood samples of all 7 patients (Fig 1). Taken together, we conclude that the dominant Vβ T cells in the peripheral blood of the SS patients are of clonal origin. Double-immunofluorescence analysis We furthermore asked whether the dominant Tcell clones in the peripheral blood of patients with SS express the CD7 antigen. Double-immunofluores-
cence staining of PBMC revealed that the percentage of circulating CD4+CD7– T cells is significantly increased compared with healthy controls (Table I). CD8+CD7– T cells were not detectable in blood samples of any of the patients (data not shown). By twocolor immunofluorescence, we analyzed whether cells of the dominant Vβ T-cell clone express the CD7 antigen. As shown in a representative analysis in Fig 2, almost all cells of the dominant Vβ clone do not express the CD7 antigen. This observation is consistent for all 7 patients tested (Table II) demonstrating that the number of clonal T cells in the peripheral blood of these patients with SS precisely matches the number of CD4+CD7– T cells. We next analyzed the coexpression of CD15s, CD26, and CD49d antigens on clonal Vβ+ T cells. As shown in Table II, the clonal Vβ+CD7– cells homogeneously express the CD15s antigen whereas the percentage of Vβ+ T cells expressing CD26 or CD49d antigen, respectively, was less than 5% in all patients tested.
DISCUSSION Several reports showed that the CD4+CD7– cell population is expanded in the blood of patients with
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Fig 1. DNA from peripheral blood lymphocytes was subjected to PCR amplification of the rearranged TCR γ chain. PCR products were analyzed by polyacrylamide gel electrophoresis. Lane 1, Size marker of DNA; lane 2, negative control (normal blood lymphocytes); lane 3, positive control (Jurkat cells); lanes 4-10, blood samples of patients with SS.
Fig 2. Representative double-immunofluorescence staining of blood lymphocytes from a patient with SS (patient 4). Peripheral blood lymphocytes were stained with anti-Vβ3 and with anti-CD7 monoclonal antibodies and analyzed by flow cytometry. Note expansion of population of Vβ3+ CD7– T cells.
SS and suggested, although not formally proved, that these cells represent the malignant cell clone.4,5 This assumption, however, has been recently called into question by the observation of clonal T cells in both the CD7+ and CD7– population of leukemic Sézary cells.14 The present study was undertaken to evaluate whether the CD4+CD7– immunophenotype is associated with cells of the dominant T-cell clone in the peripheral blood of patients with SS. We identified the dominant T-cell clone in 7 patients with SS by means of a panel of mAbs directed to var-
ious TCR Vβ regions. The detection of clonal TCR γ gene rearrangements by PCR confirmed the presence of a T-cell clone in all 7 cases. Double-immunofluorescence studies revealed that nearly all (98%) clonal TCR Vβ T cells in all 7 patients tested express the CD4+CD7– phenotype suggesting that the dominant T-cell clone originates from the CD4+CD7– Tcell subset. The results are in agreement with the finding that the CD4+CD7– T-cell subset expands in the majority of patients with CTCL.4,5,15 The data are also compatible with the observation that the num-
460 Rappl et al
ber of CD4+CD7– T cells and the number of clonal T cells identified by anti-Vβ6.7 mAbs were found identical in a patient with SS over a period of 6 months.4 Our results confirm a previous study showing that expansion of circulating CD4+CD7– cells in advanced stages of cutaneous T-cell lymphoma is associated with CD15s expression and lack of CD26 and CD49d expression.15 It remains to be determined whether the CD15s+CD26–CD49d– immunophenotype corresponds to a discrete subpopulation of mature CD4+CD7– T cells. In this context, strong expression of sialyl LewisX (CD15s) antigens on peripheral T cells has been recently observed in kidney transplant recipients with rejection indicating that CD15s expression on T cells correlates with persistent antigen stimulation in vivo.16 The following observations support the concept that the CD4+CD7– T-cell subset may represent a candidate counterpart of certain forms of CTCL: (1) The vast majority of leukemic T cells in the peripheral blood of patients with SS lack CD7 antigen expression but show high expression of cutaneous lymphocyte antigen4,5,17; (2) cutaneous infiltrates of lymphoid cells in SS and other types of CTCL contain a high number of CD4+CD7– T cells6; (3) the surface antigen CH-F42 is expressed by normal CD4+CD7– T cells and malignant CD4+CD7– Sézary cells but is absent in cells of a variety of other hematologic malignancies18; (4) physiologic CD4+CD7– T cells show a TH0/TH2-like cytokine secretion pattern and a preferential production of interleukin 5 as do malignant T cells of CTCL with TH2 activities.19-21 We therefore suggest that the very similar properties of physiologic and neoplastic CD4+CD7– T cells make the assumption unlikely that loss of CD7 expression in CTCL is the result of an unspecific antigen loss (“aberrant” phenotype) during malignant transformation.6 Our results are in apparent discrepancy to a study reported by Dummer et al14 and their general conclusion that the CD4+CD7– population does not represent the dominant T-cell clone in patients with SS. Whereas their conclusion is based on a very low number of patients, we tested circulating blood cells of 7 patients with SS by means of double-immunofluorescence analysis and consistently obtained adverse results. Our data, however, are in agreement with their recent results showing that the CD4+ CD7– immunophenotype correlates with the detection of clonal cells in the peripheral blood by PCR.22 Although a minority of patients with CD7+ SS have been described,23 absence of CD7 antigen is generally accepted to be a common feature of malignant Sézary cells. The reasons for the discrepant observations remain unresolved so far. However, it will be worthwhile to ask whether there are immunologic
J AM ACAD DERMATOL MARCH 2001
or clinical differences between the majority of SS with a CD7– phenotype and those minor cases with CD7 antigen expression. In conclusion, the present study shows that the CD4+CD7– immunophenotype is strongly associated with the dominant T-cell clone in the peripheral blood of 7 analyzed patients with SS. The results confirm and extend previous reports indicating that the flow cytometric enumeration of CD4+CD7– T cells and the analysis of expression of CD15s versus nonexpression of CD26 and CD49d antigens provides useful information for the diagnosis and follow-up in patients with SS.24-26 The results further support the concept that the majority of Sézary cells correspond to a physiologic CD4+CD7– counterpart of mature T cells. We thank S. Maas and K. Hilgert for great technical assistance, and Dr R. Gold (Würzburg) for supplying us with TCR Vβ– monoclonal antibodies. REFERENCES 1. Diamandidou E, Cohen PR, Kurzrock R. Mycosis fungoides and Sézary syndrome. Blood 1996;88:2385-409. 2. Willemze R, Sterry W, Berti E, Cerroni L, Chimenti S, Diaz-Pérez JL, et al. EORTC classification for primary cutaneous lymphoma: a proposal from the cutaneous study group of the European organisation for research and treatment of cancer. Blood 1997; 90:354-71. 3. Reinhold U, Abken H. CD4+ CD7– T cells: a separate subpopulation of memory T cells? J Clin Immunol 1997;17:265-71. 4. Bogen SA, Pelley D, Charif M, McCusher M, Koh H, Foss F, et al. Immunophenotypic identification of Sézary cells in peripheral blood. Am J Clin Pathol 1996;106:739-48. 5. Harmon CB, Witzig TE, Katzmann JA, Pittelkow MR. Detection of circulating T cells with CD4+ CD7– immunophenotype in patients with benign and malignant lymphoproliferative dermatoses. J Am Acad Dermatol 1996;35:404-10. 6. Bergman R, Faclieru D, Sahar D, Sander CA, Kerner H, Ben-Aryeh Y, et al. Immunophenotyping and T cell receptor γ gene rearrangement analysis as an adjunct to the histopathologic diagnosis of mycosis fungoides. J Am Acad Dermatol 1998;39: 554-9. 7. Moll M, Reinhold U, Kukel S, Abken H, Müller R, Oltermann I, et al. CD7 negative helper T cells accumulate in inflammatory skin lesions. J Invest Dermatol 1994;102:328-32. 8. Reinhold U, Abken H, Kukel S, Moll M, Oltermann I, Müller R, et al. CD7– T cells represent a subset of normal human blood lymphocytes. J Immunol 1993;150:2081-9. 9. Reinhold U, Liu L, Sesterhenn J, Abken H. CD7-negative T cells represent a separate differentiation pathway in a subset of post-thymic helper T cells. Immunology 1996;89:391-6. 10. Foster CA,Yokozeki H, Rappersberger K, Koning F,Volc-Platzer B, Rieger A, et al. Human epidermal T cells predominantly belong to the lineage expressing alpha/beta T cell receptor. J Exp Med 1990;171:997-1013. 11. Davis AL, McKenzie JL, Hart DN. HLA-DR–positive leucocyte subpopulations in human skin include dendritic cells, macrophages, and CD7-negative T cells. Immunology 1988;65: 573-81. 12. Muche JM, Lukowsky A, Asadullah K, Gellrich S, Sterry W. Demonstration of frequent occurrence of clonal T cells in the
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20. Autran B, Legac E, Blanc C, Debré P. A Th0/Th2-1ike function of CD4+ CD7– T helper cells from normal donors and HIV-infected patients. J Immunol 1995;154:1408-17. 21. Dummer R, Heald PW, Nestle FO, Ludwig E, Laine E, Hemmi S, et al. Sézary syndrome’s T cell clones display T helper 2 cytokines and express the accessory factor-1 (interferon gamma receptor beta chain). Blood 1996;88:1383-9. 22. Laetsch B, Haffner AC, Dobbeling U, Seifert B, Ludwig E, Burg G, et al. CD4+/CD7– T cell frequency and polymerase chain reaction-based clonality assay correlate with stage in cutaneous T cell lymphomas. J Invest Dermatol 2000;114:107-11. 23. Yagi H, Tokura Y, Furukawa F, Takigawa M. CD7-positive Sézary syndrome with a Th1 cytokine profile. J Am Acad Dermatol 1996;34:368-74. 24. Fritz TM, Kleinhans M, Nestle FO, Burg G, Dummer R. Combination treatment with extracorporeal photopheresis, interferon alfa and interleukin-2 in a patient with the Sézary syndrome. Br J Dermatol 1999;140:1144-7. 25. Bernengo MG, Quaglino P, Novelli M, Cappello N, Doveil GC, Lisa F, et al. Prognostic factors in Sézary syndrome: a multivariate analysis of clinical, haematological and immunological features. Ann Oncol 1998;9:857-63. 26. Collenberg C, von den Driesch P, Gramatzki M, Fartasch M, Gruschwitz MS. Monitoring of the disease progress in Sézary syndrome by CD7– cells using flow cytometry. Br J Dermatol 1996;134:980-2.
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