- Email: [email protected]
inducer T cells (CD45RA-, CDw29+)
CD4+ Cutaneous T-Cell Lymphomas Show the Phenotype of Helper/Inducer T Cells (CD45RA-, CDw29+) Wolfram
Sterry, M.D. and Volker Mielke,
M.D.
Department of Dermatology, University of Kiel, Kiel, Federal Republic of Germany.
blood both subsets are equally distributed, we present evidence that all CD4+ cutaneous T-cell lymphomas are of the helper/inducer T cell p h enotype. These findings are of importance both for pathogenetic and clinical considerations: the presence of plasma cells in dermal infiltrates and the elevation of serum immunoglobulins in patients of mycosis fungoides may be the consequence of interleukin-4 secretion of the neoplastic CD4+ helper/inducer cells. The exclusive memory T cell phenotype of cutaneous T-cell lymphomas may be due to a general predominance of this subset in the skin, or be the consequence of cellular activation during malignant transformation.] Invest Dermato/ 93:413 - 4 16, 2 989
CD4+ T cells are heterogenous and include at least two subsets that differ in their influence to immunoglobin synthesis, cytokine secretion pattern and immunophenotype. Among others these subsets have been designated as suppressor/inducer or naive T cells (CD45RA+, CDw29-) and helper/inducer or memory T cells (CD45RA-, CDw29+). Current theories suggest that these CD4+ T-cell subsets either reflect sequential stages of maturation before and after activation (antigen contact) or represent distinct lineages. In this study, we systematically analyzed the participation of both suppressor/inducer (CD45RA+) and helper/inducer (CDw29+) T cells in the dermal lymphohistiocytic infiltrate of various CD4+ cutaneous T-cell lymphomas. Although in peripheral
H
uman T cells are categorized
into two general subsets, i.e., helper and suppressor cells, based on their expression of CD4 and CD8 molecules, respectively [I]. It has become evident that T cells expressing the CD4 molecule are heterogenous, and include a suppressor/inducer as well as a helper/inducer subpopulation [2,3]. This evidence arose by studies using cloned CD4+ T-cell lines as well as CD4+ subpopulations separated by certain surface molecules, particularly those recognized by the antibodies 2H4 and 4B4 [2 - 41. These CD4+ subsets differ in their influence on immunoglobin synthesis, proliferative response to various stimuli, pattern of secreted cytokines, and surface phenotype [5]. At present, different nomenclatures are used to designate these subsets; those most frequently used are suppressor/inducer, naive, or inflammatory T cells on the one hand, and helper/inducer, memory, or helper T cells on the other [2,3,5,6]. In this study we use the terms suppressor/inducer and helper/inducer T cells.
Suppressor/inducer T cells are able to inhibit the pokeweed mitogen (PWM)-driven immunoglobulin synthesis of B cells [2]. They proliferate to concanavalin A (ConA) stimulation, a mitogen well known to induce suppressor activity, but poorly to soluble antigens [2]. Although they are good producers of interleukin-2 (IL-~) and probably (INF-y), they do not secrete IL-4; remarkably, only this subset is able to produce lymphotoxin (TNF-B). This suppressor/inducer CD4+ subset is characterized by the CD45RA antigen, which represents an epitope on the leukocyte common antigen (LCA) family. The LCA (CD45) family, which consists of at least six isoforms (180 to 240 kd glycoproteins), is expressed exclusively on cells of hemopoietic origin. The LCA gene uses about 30 exons, and the various isoforms are generated by differential splicing of the exons 4, 5, and 6 [7,8]; in consequence, LCA isoforms have a shared and a variable segment. Antigenic determinants common to all isoforms have been grouped together as CD45 antigens [9]. Depending on the leukocyte cell type, various numbers and combinations of isoforms are expressed [lo], epitopes of the variable part will be restricted to distinct leukocyte sub opulations; such epitopes are clustered as CD45R antigens (R Por restricted) [9]. It has been shown recently that the CD45RA antigen is encoded by exon 4 (also called exon A) of the LCA gene [ 1 l]. Analyzing these glycoproteins on CD4+ T cells, it became evident that helper/inducer T cells express at least four LCA isoforms (180, 190, 200, and 220 kd), whereas suppressor/inducer T cells synthesize only the 180- and I9O-kd isoforms. The CD45RA antigen, hallmark of the suppressor/inducer T cells, is present only on the 200- and 22O-kd isoform, anti-CD45RA antibodies are able to abrogate the specific suppressor/inducer function of this subset [ 121. In contrast, helper/inducer CD4+ T cells are capable of inducing both polyclonal immunoglobulin synthesis of B cells in PWM driven assays and antigen-specific immunoglobin production [3]. They are readily activated upon various stimuli, including soluble antigen. Activation leads to secretion of large amounts of IL-4 [5],
Manuscript received August 23,1988; accepted for publication March 20, 1989. This investigation was supported by the DFG, Deutsche Forschungsgemeinschaft, grant Ste 366/2-l. Reprint requests to: Wolfram Sterry, M.D., Department of Dermatology, University of Kiel, Schittenhelmstrabe 7.2300 Kiel 1, Federal Republic of Germany. Abbreviations: ConA: concanavalin A IL-2: interleukin-2 LCA: leukocyte common antigen PBS: phosphate-buffered saline PWM: pokeweed mitogen VLA: very late antigen
0022-202X/89/$03.50
Copyright 0 1989 by The Society for Investigative Dermatology, Inc. 413
414
STERRY
AND
MIELKE
THE JOURNAL
TABLE I. Cellular Infiltrate From Various CD4+ Cutaneous T-Cell Lymphomas Analyzed Immunophenotypically Using a Panel of Monoclonal Antibodies Ag Designation
mAb Used
CD2 CD3 CD5 CD4
Let@ Leu4” Leul” Leu3’
CD8 CD45RO
LeU2’ UCHLI”
CD45RA
LeulB”
CDw29
4Be
Ag Distribution AI1 T cells All T cells Most T cells, some B cells Suppressor/inducer T cells, helper/inducer T cell, activated macrophages Suppressor/cytotoxic T cells T cells; 30-fold higher expressed on hle per /‘dm ucer as on suppressor/inducer T cells, minority of monocytes CD4+ suppressor/inducer T cells, B cells (Leul8 detects the same antigen as 2H4 antibody; see Ref 2) CD4+ helper/inducer T cells, keratinocytes, broad variety of soft tissues
a Becton Dickinson, Heidelberg, F.R.G. b Dakopatts, Hamburg, F.R.G.
cCoulter
Electronics,
Krefeld, F.R.G.
OF INVESTIGATIVE
DERMATOLOGY
which supports the synthesis of antigen specific immunoglobulin as well as switch from IgG to IgE immunoglobulin subclass. The characterizing surface molecule of the helper/inducer subset is the CDw29 antigen, which probably belongs to the so-called very late antigen (VLA) family of the integrin superfamily of adhesion molecules [4,13]. It is of interest that the helper/inducer subset expresses several other adhesion molecules, such as LFA-3, CD2, and LFA-1 [14], in high amounts. It is remarkable that only the low molecular weight CD45 isoform (CD45R0, UCHLI) shows a high density on helper/inducer cells. Despite its obvious importance only fragmentary data are available concerning the participation of both subsets in peripheral CD4+ T-cell lymphomas [15]. The lack of these studies is particularly due to the fact that the CD45RA antigen is also expressed on B cells, and thus difficulties arise in the analysis of infiltrates rich of B cells, for example, in lymph nodes. As B cells are either absent or extremely rare in cutaneous CD4+ T-cell lymphomas [ 161, we systematically analysed the dermal lymphohistiocyte infiltrates in different cutaneous CD4+ T-cell lymphomas. We present evidence that virtually all of these cutaneous malignant CD4+ T-cell infiltrates carry the phenotype of helper/inducer T cells.
TABLE II. Lmmunophenotype
CD2
CD3
CD5
CD4
CD8
Mycosis fungoides 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
+ + + + + + + + + + + + + + +
•l+ + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
_ -
+ i+ + + + + + + + f + + + +
_ _ _
+ + + + + + + + + + + + + + +
Sezary syndrome 16. 17.
+ +
+ +
+ +
+ +
-
+ +
_ -
+ +
-
Parapsoriasis plaques 18. 19. 20. 21. 22.
+ + + + +
+ + + + +
+ + + + +
+ + + + +
_ -
+ + + + +
_ -
+ + + + +
-
Lymphomatoid papulosis 23. 24. 25. 26.
+ + + +
+ + + +
+ + + +
+ + + +
-
+ + + +
-
+ + + +
-
Pleomorphic T-cell lymphoma 27. 28. 29. 30.
+ + + +
+ + + +
+ + + +
+ + + +
-
t + + +
_ -
+ + + +
-
High-grade lymphomas 31. 32. 33.
+ + +
+ + +
+ + +
+ + +
-
+ + +
-
+ + +
-
CD45RO
CD45RA
CDw29
CD22
-
-
en
T-cell
Cases with less than 15% positive cells have been classified as negative
for the respective
antigen:
positive if more than 80% of cells were labeled.
-
-
VOL. 93. NO. 3
SEPTEMBER
MATERIALS
D4+ T CELLS
1989
AND
415
METHODS
The cellular infiltrate from various CD4+ cutaneous T-cell lymphomas was analyzed immunophenotypically using a panel of monoclonal antibodies (Table I): mycosis fungoides (n = 15), Sezary syndrome (n = 2), pleomorphic T-cell lymphoma (n = 5), high-grade malignant T-cell lymphoma of the skin (n = 3), lymphomatoid papulosis (n = 4), and parapsoriasis en plaques (n = 5). In this study, we used the classification of Suchi and co-workers [IV* From each case immunohistochemical stainings were performed on 6-pm cryostat sections. Using a three-step immunoperoxidase technique, samples were sequentially incubated at room temperature with the primary monoclonal antibody, peroxidase conjugated rabbit antimouse IgG [Dakopatts, Hamburg, F.R.G.; diluted 1: 15 with human serum/phosphate-buffered saline (PBS)], and thereafter with peroxidase-conjugated goat antirabbit IgG (Medac, Hamburg, F.R.G.; diluted 1: I5 with human serum/PBS). After each step a brief rinse in NaCl/Tris buffer followed. Peroxidase activity was visualized with 0.06% diaminobenzidine (Walter, Kiel, with F.R.G.) and 0.01% HaOa [18]. S ec t ions were counterstained hemalaun and mounted with glycerin gelatin. In each section, 200 cells of the lymphohistiocytic infiltrate were analyzed; the numbers of cells expressing the respective antigen are given in percent. RESULTS All analyzed CD4+T-cell lymphomas, both of low- and high-grade malignancy, showed the phenotypic profile of helper/inducer T cells: CD2+, CD3+, CD5+, CD4+, CD45RO+, CD45RA-, CDw29+ (Table II, Fig 1). The same was true for lymphomatoid papulosis and parapsoriasis en plaques. Suppressor/inducer CD45RA+ T cells were found scattered within these infiltrates, ranging from 2 to 15% of the infiltrate cells (Table II, Fig 2). In all infiltrates B cells, which are CD45RA+, were either absent or rare (less than 3% CD22+). In contrast to the CD45R antigens (CD45RO/UCHLl, CD45RA/Leul8), ex p ression of CDw29 was not confined to the infiltrate cells, but was also found on other cell types, particularly endothelial cells, fibroblasts, cutaneous nerves, and smooth muscle cells. The epidermis in normal and diseased skin showed constant CDw29 expression on basal cells. DISCUSSION It has already been demonstrated more than ten years ago that the leukemic tumor cells from SCzary syndrome are able to provide help for immunoglobulin synthesis [I9]. In contrast, the assessment of the functional capacity of T cells infiltrating the skin in nonleukemic cutaneous T-cell lymphomas is greatly hampered by the difficulties in isolating them from the tight collagen network of the dermis; therefore, no studies concerning this problem have been undertaken so far. The results presented in this study provide phenotypical evidence that the great majority of CD4+ lymphocytes infiltrating the skin in mycosis fungoides, other cutaneous T-cell lymphomas, and related disorders belong to the functional subgroup of helper/inducer CD45RA-/CDw29+ T cells. This contrasts with the subset distribution in peripheral blood, where only about 40% of T cells express this phenotype; in healthy adults, the CDw29 antigen is present on about 40 to 50% of peripheral CD4+ T cells, whereas the CD45RA antigen is found on 50 to 60% of peripheral CD4+T cells [3,20,21]. At present, there are two mutually exclusive concepts describing the relationship between the two CD4+ subsets. Sanders and coworkers suggested that CD4+ T cells exhibit a different phenotype before (naive) and after (memory) their first activation [5,22], based on observations on human cord lymphocytes. These cord blood lymphocytes, which express the CD45RA antigen, show after polyclonal activation a permanent increase in the expression of CDw29, LFA-3, CD2, and LFA-1, as well as of the CD45RO antigen; in contrast, the high CD45RA expression is profoundly re-
Figure 1. CDw29 expression on infiltrate cells and on keratinocytes in mycosis fungoides, plaque stage. line: dermoepidermal junction.
duced [5,141. This irreversible change of the phenotype induced by first stimulation may be biologically important, as these memory (h e lp er /’m d ucer ) ce 11s are readily activated on repeated antigen contact, making them more effective in eliciting an effective immune response to antigens to which the individual already has been exposed. Assuming that this concept describes the actual situation, this would imply that T cells that undergo malignant degeneration in cutaneous CD4+ T-cell lymphomas have been antigen stimulated. One could conclude that these diseases arise during an uneffective response to yet unidentified antigens; this view would be supported by the fact that the same helper/inducer predominance is also found in parapsoriasis en plaques, a precursor disease of mycosis fungoides. A second concept describing the relationship between the CD4+ subsets postulates the existence of two distinct lineages, as proposed by Janeway and co-workers [4], on the basis of their extensive studies with cloned CD4+ ceil lines. It is questionable, however, whether subsets in cloned cells, which actually represent chronically stimulated T cells, can be compared with the naive and memory subsets. The fact that the malignant cells in different CD4+ cutaneous T-cell lymphomas phenotypically belong to the helper/inducer Tcell subset may explain several clinical observations. First, the plasma cells nests, which are regularly found in mycosis fungoides and pleomorphic T-cell lymphoma, may be induced by the CD45RA-/CDw29+ tumor cells either through direct cell contacts or by secretion of cytokines, which induce plasma cell differentia-
416
STERRY
AND MIELKE
THE JOURNAL
human 1985b
helper inducer
OF INVESTIGATIVE
T cell subset. J Immunol
DERMATOLOGY
134:3762-
3767,
Janeway CA, Carding JRS, Jones B, Murray J, Portoles P, Rasmussen R, Rojo J, Saizawa K, West J, Bottomly K: CD4+ T cells: specificity and function. Immunol Rev 101:39-80, 1988 Sanders ME, Makgoba MW, Shaw S: Human naive and memory T cells: reinterpretation of helper-inducer and suppressor-inducer subsets. Immunol Today 9:195- 198, 1988a Bottomly K: A functional dichotomy munol Today 9:268 - 274, 1988
in CD4+ T lymphocytes.
Im-
Streuli M, Hall LR, Saga Y, Schlossman SF, Saito H: Differential usage of three exons generates at least five different mRNAs encoding human leucocyte common antigens. J Exp Med 166:15481566, 1987b
Figure 2. Few CD45RA positive cells in an infiltrate line: dermoepidermal junction.
ofmycosis
fungoides.
tion, especially by IL-4. In this context it should the presence of such plasma cells in the dermal
be mentioned that has been infiltrate associated with an unfavorable clinical prognosis [23]. Second, the frequent elevation of immunoglobulins in the sera of patients with mycosis fungoides [16] may be induced by the tumor cells. The exclusive memory T-cell phenotype of cutaneous T-cell lymphomas may be due to a general predominance of this subset in the skin, or be the consequence of cellular activation during malignant transformation. Work is in progress to clarify if a predominance of helper/inducer T ceils does also occur in reactive skin diseases, and whether the transition of naive into memory T cells
takes place extracutaneously
or within
8.
Thomas ML, Lefrancois: Differential expression of the leucocyte mon antigen family. Immunol Today 9:320-326, 1988
9.
Cobbold S, Hale G, Waldmann H: Non-lineage, LFA-1 family, and leucocyte common antigens: new and previously defined clusters. In: McMichael AJ (ed). Leucocyce Typing, White Cell Differentiation Antigens. New York, Oxford, 1987, pp 788-803
10.
Rudd CE, Morimoto C, Wong LL, Schlossman SF: The subdivision the T4 (CD4) subset on the basis of the differential expression L-C/T200 antigens. J Exp Med 166:17581773, 1987a
11.
Streuli M, Matsuyama T, Morimoto C, Schlossman SF, Saito H: Identification of the sequence required for expression of the 2H4 epitope on the human leukocyte common antigens. J Exp Med 166:15671572, 1987a
12.
Rudd CE, Morimoto C, Wong LL, Schlossman SF: The structure of the 2H4 antigen: a subset of the LCA/T200 family of antigens involved in immune suppression. In: McMichael AJ (ed). Leucocyte Typing, White Cell Differentiation Antigens. New York, Oxford, 1987, pp 242-245
13.
Ruoslahti E, Pierschbacher MD: New perspectives in cell adhesion: RGD and integrins. Science 238:491-497, 1987
14.
Sanders ME, Makgoba MW, Sharrow SO, Stephany D, Springer TA, Young HA, Shaw S: Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-1) and three other molecules (UCHLI, CDw29, and Pgp-1) and have enhanced IFN-gamma production. J 1mmuno1140:14011407, 1988a
15.
Ralfkiaer E, Lange Wantzin G: HTLV-positive and -negative cutaneous T-cell lymphomas in Denmark. An immunohistochemical study of T-cell antigens and T-cell subset associated antigens. In: McMichael AJ (ed). Leucocyte Typing, White Cell Differentiation Antigens. New York, Oxford, 1987, pp 290-291
16.
Sterry W: Mycosis fungoides. In: Berry CL (ed). Dermatopathology. Berlin, Springer, 1985, pp 167 - 223
17.
Suchi T, Lennert K, Tu LY, Kikuchi M, Sato E, Stansfeld AG, Feller AC: Histopathology and immunohistochemistry of peripheral Tcell lymphomas - a proposal for classification of T-cell lymphomas. Clin Path 40:995- 1015, 1987
18.
Stein H, Gerdes J, Schwab U, Lemke H, Mason DY, Ziegler A, Schienle W, Diehl V: Identification of Hodgkin and Sternberg Reed cells as a unique cell type derived from a newly detected small cell population. Int J Cancer 30:445 - 449, 1982
19.
Broder S, Edelson RL, Lutzner MA, Nelson DL, MacDermott RP, Durm ME, Goldman CK, Meade BD, Waldmann TA: The Sezary syndrome. A malignant proliferation of helper T cells. J Clin Invest 58:1297-1306, 1976
20.
Smith SH, Brown MH, Rowe D, Callard RE, Beverly PCL: Functional subsets of human helper-inducer cells defined by a new monoclonal antibody, UCHLI. Immunology 58:63-70, 1986
21.
De Paoli P, Battistin
the skin.
The excellent technical assistance of Miss S. Heimrich is gratefully acknowledged.
lymphocyte subsets: (suppressor inducer) 48:290 - 296, 1988
REFERENCES Romain PL, Schlossman Invest 74:1559-1565,
SF: Human 1984
T lymphocyte
subsets. J Clin
Morimoto C. Letvin NL, Discaso JA, Aldrich WR, Schlossman SF: The isolation and characterization of the human suppressor inducer T cell sub%et. J Immunol 134:1508-1514, 1985a Morimoto C, Letvin NL, Boyd AW, HaganM, Brown HM, Kornacki MM, Schlossman SF: The isolation and characterization of the
com-
of of
S, Santini GF: Age-related changes in human progressive reduction of the CD4 CD45R population. Clin Immunol Immunopathol
22.
Serra HM, Krowka JF, Ledbetter (Lp220) represents a post-thymic munol 140:1435-1441, 1988
23.
Chimenti S, Hoed1 S, Smolle J, Soyer HP, Torne R, Kerl H: Prognostic significance of plasma cell infiltrates in cutaneous T cell lymphomas. Am J Dermatopathol 9:167, 1987
JA, Pilarski LM: Loss of CD45R T cell differentiation event. J Im-
Sterry, M.D. and Volker Mielke,
M.D.
Department of Dermatology, University of Kiel, Kiel, Federal Republic of Germany.
blood both subsets are equally distributed, we present evidence that all CD4+ cutaneous T-cell lymphomas are of the helper/inducer T cell p h enotype. These findings are of importance both for pathogenetic and clinical considerations: the presence of plasma cells in dermal infiltrates and the elevation of serum immunoglobulins in patients of mycosis fungoides may be the consequence of interleukin-4 secretion of the neoplastic CD4+ helper/inducer cells. The exclusive memory T cell phenotype of cutaneous T-cell lymphomas may be due to a general predominance of this subset in the skin, or be the consequence of cellular activation during malignant transformation.] Invest Dermato/ 93:413 - 4 16, 2 989
CD4+ T cells are heterogenous and include at least two subsets that differ in their influence to immunoglobin synthesis, cytokine secretion pattern and immunophenotype. Among others these subsets have been designated as suppressor/inducer or naive T cells (CD45RA+, CDw29-) and helper/inducer or memory T cells (CD45RA-, CDw29+). Current theories suggest that these CD4+ T-cell subsets either reflect sequential stages of maturation before and after activation (antigen contact) or represent distinct lineages. In this study, we systematically analyzed the participation of both suppressor/inducer (CD45RA+) and helper/inducer (CDw29+) T cells in the dermal lymphohistiocytic infiltrate of various CD4+ cutaneous T-cell lymphomas. Although in peripheral
H
uman T cells are categorized
into two general subsets, i.e., helper and suppressor cells, based on their expression of CD4 and CD8 molecules, respectively [I]. It has become evident that T cells expressing the CD4 molecule are heterogenous, and include a suppressor/inducer as well as a helper/inducer subpopulation [2,3]. This evidence arose by studies using cloned CD4+ T-cell lines as well as CD4+ subpopulations separated by certain surface molecules, particularly those recognized by the antibodies 2H4 and 4B4 [2 - 41. These CD4+ subsets differ in their influence on immunoglobin synthesis, proliferative response to various stimuli, pattern of secreted cytokines, and surface phenotype [5]. At present, different nomenclatures are used to designate these subsets; those most frequently used are suppressor/inducer, naive, or inflammatory T cells on the one hand, and helper/inducer, memory, or helper T cells on the other [2,3,5,6]. In this study we use the terms suppressor/inducer and helper/inducer T cells.
Suppressor/inducer T cells are able to inhibit the pokeweed mitogen (PWM)-driven immunoglobulin synthesis of B cells [2]. They proliferate to concanavalin A (ConA) stimulation, a mitogen well known to induce suppressor activity, but poorly to soluble antigens [2]. Although they are good producers of interleukin-2 (IL-~) and probably (INF-y), they do not secrete IL-4; remarkably, only this subset is able to produce lymphotoxin (TNF-B). This suppressor/inducer CD4+ subset is characterized by the CD45RA antigen, which represents an epitope on the leukocyte common antigen (LCA) family. The LCA (CD45) family, which consists of at least six isoforms (180 to 240 kd glycoproteins), is expressed exclusively on cells of hemopoietic origin. The LCA gene uses about 30 exons, and the various isoforms are generated by differential splicing of the exons 4, 5, and 6 [7,8]; in consequence, LCA isoforms have a shared and a variable segment. Antigenic determinants common to all isoforms have been grouped together as CD45 antigens [9]. Depending on the leukocyte cell type, various numbers and combinations of isoforms are expressed [lo], epitopes of the variable part will be restricted to distinct leukocyte sub opulations; such epitopes are clustered as CD45R antigens (R Por restricted) [9]. It has been shown recently that the CD45RA antigen is encoded by exon 4 (also called exon A) of the LCA gene [ 1 l]. Analyzing these glycoproteins on CD4+ T cells, it became evident that helper/inducer T cells express at least four LCA isoforms (180, 190, 200, and 220 kd), whereas suppressor/inducer T cells synthesize only the 180- and I9O-kd isoforms. The CD45RA antigen, hallmark of the suppressor/inducer T cells, is present only on the 200- and 22O-kd isoform, anti-CD45RA antibodies are able to abrogate the specific suppressor/inducer function of this subset [ 121. In contrast, helper/inducer CD4+ T cells are capable of inducing both polyclonal immunoglobulin synthesis of B cells in PWM driven assays and antigen-specific immunoglobin production [3]. They are readily activated upon various stimuli, including soluble antigen. Activation leads to secretion of large amounts of IL-4 [5],
Manuscript received August 23,1988; accepted for publication March 20, 1989. This investigation was supported by the DFG, Deutsche Forschungsgemeinschaft, grant Ste 366/2-l. Reprint requests to: Wolfram Sterry, M.D., Department of Dermatology, University of Kiel, Schittenhelmstrabe 7.2300 Kiel 1, Federal Republic of Germany. Abbreviations: ConA: concanavalin A IL-2: interleukin-2 LCA: leukocyte common antigen PBS: phosphate-buffered saline PWM: pokeweed mitogen VLA: very late antigen
0022-202X/89/$03.50
Copyright 0 1989 by The Society for Investigative Dermatology, Inc. 413
414
STERRY
AND
MIELKE
THE JOURNAL
TABLE I. Cellular Infiltrate From Various CD4+ Cutaneous T-Cell Lymphomas Analyzed Immunophenotypically Using a Panel of Monoclonal Antibodies Ag Designation
mAb Used
CD2 CD3 CD5 CD4
Let@ Leu4” Leul” Leu3’
CD8 CD45RO
LeU2’ UCHLI”
CD45RA
LeulB”
CDw29
4Be
Ag Distribution AI1 T cells All T cells Most T cells, some B cells Suppressor/inducer T cells, helper/inducer T cell, activated macrophages Suppressor/cytotoxic T cells T cells; 30-fold higher expressed on hle per /‘dm ucer as on suppressor/inducer T cells, minority of monocytes CD4+ suppressor/inducer T cells, B cells (Leul8 detects the same antigen as 2H4 antibody; see Ref 2) CD4+ helper/inducer T cells, keratinocytes, broad variety of soft tissues
a Becton Dickinson, Heidelberg, F.R.G. b Dakopatts, Hamburg, F.R.G.
cCoulter
Electronics,
Krefeld, F.R.G.
OF INVESTIGATIVE
DERMATOLOGY
which supports the synthesis of antigen specific immunoglobulin as well as switch from IgG to IgE immunoglobulin subclass. The characterizing surface molecule of the helper/inducer subset is the CDw29 antigen, which probably belongs to the so-called very late antigen (VLA) family of the integrin superfamily of adhesion molecules [4,13]. It is of interest that the helper/inducer subset expresses several other adhesion molecules, such as LFA-3, CD2, and LFA-1 [14], in high amounts. It is remarkable that only the low molecular weight CD45 isoform (CD45R0, UCHLI) shows a high density on helper/inducer cells. Despite its obvious importance only fragmentary data are available concerning the participation of both subsets in peripheral CD4+ T-cell lymphomas [15]. The lack of these studies is particularly due to the fact that the CD45RA antigen is also expressed on B cells, and thus difficulties arise in the analysis of infiltrates rich of B cells, for example, in lymph nodes. As B cells are either absent or extremely rare in cutaneous CD4+ T-cell lymphomas [ 161, we systematically analysed the dermal lymphohistiocyte infiltrates in different cutaneous CD4+ T-cell lymphomas. We present evidence that virtually all of these cutaneous malignant CD4+ T-cell infiltrates carry the phenotype of helper/inducer T cells.
TABLE II. Lmmunophenotype
CD2
CD3
CD5
CD4
CD8
Mycosis fungoides 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
+ + + + + + + + + + + + + + +
•l+ + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + +
_ -
+ i+ + + + + + + + f + + + +
_ _ _
+ + + + + + + + + + + + + + +
Sezary syndrome 16. 17.
+ +
+ +
+ +
+ +
-
+ +
_ -
+ +
-
Parapsoriasis plaques 18. 19. 20. 21. 22.
+ + + + +
+ + + + +
+ + + + +
+ + + + +
_ -
+ + + + +
_ -
+ + + + +
-
Lymphomatoid papulosis 23. 24. 25. 26.
+ + + +
+ + + +
+ + + +
+ + + +
-
+ + + +
-
+ + + +
-
Pleomorphic T-cell lymphoma 27. 28. 29. 30.
+ + + +
+ + + +
+ + + +
+ + + +
-
t + + +
_ -
+ + + +
-
High-grade lymphomas 31. 32. 33.
+ + +
+ + +
+ + +
+ + +
-
+ + +
-
+ + +
-
CD45RO
CD45RA
CDw29
CD22
-
-
en
T-cell
Cases with less than 15% positive cells have been classified as negative
for the respective
antigen:
positive if more than 80% of cells were labeled.
-
-
VOL. 93. NO. 3
SEPTEMBER
MATERIALS
D4+ T CELLS
1989
AND
415
METHODS
The cellular infiltrate from various CD4+ cutaneous T-cell lymphomas was analyzed immunophenotypically using a panel of monoclonal antibodies (Table I): mycosis fungoides (n = 15), Sezary syndrome (n = 2), pleomorphic T-cell lymphoma (n = 5), high-grade malignant T-cell lymphoma of the skin (n = 3), lymphomatoid papulosis (n = 4), and parapsoriasis en plaques (n = 5). In this study, we used the classification of Suchi and co-workers [IV* From each case immunohistochemical stainings were performed on 6-pm cryostat sections. Using a three-step immunoperoxidase technique, samples were sequentially incubated at room temperature with the primary monoclonal antibody, peroxidase conjugated rabbit antimouse IgG [Dakopatts, Hamburg, F.R.G.; diluted 1: 15 with human serum/phosphate-buffered saline (PBS)], and thereafter with peroxidase-conjugated goat antirabbit IgG (Medac, Hamburg, F.R.G.; diluted 1: I5 with human serum/PBS). After each step a brief rinse in NaCl/Tris buffer followed. Peroxidase activity was visualized with 0.06% diaminobenzidine (Walter, Kiel, with F.R.G.) and 0.01% HaOa [18]. S ec t ions were counterstained hemalaun and mounted with glycerin gelatin. In each section, 200 cells of the lymphohistiocytic infiltrate were analyzed; the numbers of cells expressing the respective antigen are given in percent. RESULTS All analyzed CD4+T-cell lymphomas, both of low- and high-grade malignancy, showed the phenotypic profile of helper/inducer T cells: CD2+, CD3+, CD5+, CD4+, CD45RO+, CD45RA-, CDw29+ (Table II, Fig 1). The same was true for lymphomatoid papulosis and parapsoriasis en plaques. Suppressor/inducer CD45RA+ T cells were found scattered within these infiltrates, ranging from 2 to 15% of the infiltrate cells (Table II, Fig 2). In all infiltrates B cells, which are CD45RA+, were either absent or rare (less than 3% CD22+). In contrast to the CD45R antigens (CD45RO/UCHLl, CD45RA/Leul8), ex p ression of CDw29 was not confined to the infiltrate cells, but was also found on other cell types, particularly endothelial cells, fibroblasts, cutaneous nerves, and smooth muscle cells. The epidermis in normal and diseased skin showed constant CDw29 expression on basal cells. DISCUSSION It has already been demonstrated more than ten years ago that the leukemic tumor cells from SCzary syndrome are able to provide help for immunoglobulin synthesis [I9]. In contrast, the assessment of the functional capacity of T cells infiltrating the skin in nonleukemic cutaneous T-cell lymphomas is greatly hampered by the difficulties in isolating them from the tight collagen network of the dermis; therefore, no studies concerning this problem have been undertaken so far. The results presented in this study provide phenotypical evidence that the great majority of CD4+ lymphocytes infiltrating the skin in mycosis fungoides, other cutaneous T-cell lymphomas, and related disorders belong to the functional subgroup of helper/inducer CD45RA-/CDw29+ T cells. This contrasts with the subset distribution in peripheral blood, where only about 40% of T cells express this phenotype; in healthy adults, the CDw29 antigen is present on about 40 to 50% of peripheral CD4+ T cells, whereas the CD45RA antigen is found on 50 to 60% of peripheral CD4+T cells [3,20,21]. At present, there are two mutually exclusive concepts describing the relationship between the two CD4+ subsets. Sanders and coworkers suggested that CD4+ T cells exhibit a different phenotype before (naive) and after (memory) their first activation [5,22], based on observations on human cord lymphocytes. These cord blood lymphocytes, which express the CD45RA antigen, show after polyclonal activation a permanent increase in the expression of CDw29, LFA-3, CD2, and LFA-1, as well as of the CD45RO antigen; in contrast, the high CD45RA expression is profoundly re-
Figure 1. CDw29 expression on infiltrate cells and on keratinocytes in mycosis fungoides, plaque stage. line: dermoepidermal junction.
duced [5,141. This irreversible change of the phenotype induced by first stimulation may be biologically important, as these memory (h e lp er /’m d ucer ) ce 11s are readily activated on repeated antigen contact, making them more effective in eliciting an effective immune response to antigens to which the individual already has been exposed. Assuming that this concept describes the actual situation, this would imply that T cells that undergo malignant degeneration in cutaneous CD4+ T-cell lymphomas have been antigen stimulated. One could conclude that these diseases arise during an uneffective response to yet unidentified antigens; this view would be supported by the fact that the same helper/inducer predominance is also found in parapsoriasis en plaques, a precursor disease of mycosis fungoides. A second concept describing the relationship between the CD4+ subsets postulates the existence of two distinct lineages, as proposed by Janeway and co-workers [4], on the basis of their extensive studies with cloned CD4+ ceil lines. It is questionable, however, whether subsets in cloned cells, which actually represent chronically stimulated T cells, can be compared with the naive and memory subsets. The fact that the malignant cells in different CD4+ cutaneous T-cell lymphomas phenotypically belong to the helper/inducer Tcell subset may explain several clinical observations. First, the plasma cells nests, which are regularly found in mycosis fungoides and pleomorphic T-cell lymphoma, may be induced by the CD45RA-/CDw29+ tumor cells either through direct cell contacts or by secretion of cytokines, which induce plasma cell differentia-
416
STERRY
AND MIELKE
THE JOURNAL
human 1985b
helper inducer
OF INVESTIGATIVE
T cell subset. J Immunol
DERMATOLOGY
134:3762-
3767,
Janeway CA, Carding JRS, Jones B, Murray J, Portoles P, Rasmussen R, Rojo J, Saizawa K, West J, Bottomly K: CD4+ T cells: specificity and function. Immunol Rev 101:39-80, 1988 Sanders ME, Makgoba MW, Shaw S: Human naive and memory T cells: reinterpretation of helper-inducer and suppressor-inducer subsets. Immunol Today 9:195- 198, 1988a Bottomly K: A functional dichotomy munol Today 9:268 - 274, 1988
in CD4+ T lymphocytes.
Im-
Streuli M, Hall LR, Saga Y, Schlossman SF, Saito H: Differential usage of three exons generates at least five different mRNAs encoding human leucocyte common antigens. J Exp Med 166:15481566, 1987b
Figure 2. Few CD45RA positive cells in an infiltrate line: dermoepidermal junction.
ofmycosis
fungoides.
tion, especially by IL-4. In this context it should the presence of such plasma cells in the dermal
be mentioned that has been infiltrate associated with an unfavorable clinical prognosis [23]. Second, the frequent elevation of immunoglobulins in the sera of patients with mycosis fungoides [16] may be induced by the tumor cells. The exclusive memory T-cell phenotype of cutaneous T-cell lymphomas may be due to a general predominance of this subset in the skin, or be the consequence of cellular activation during malignant transformation. Work is in progress to clarify if a predominance of helper/inducer T ceils does also occur in reactive skin diseases, and whether the transition of naive into memory T cells
takes place extracutaneously
or within
8.
Thomas ML, Lefrancois: Differential expression of the leucocyte mon antigen family. Immunol Today 9:320-326, 1988
9.
Cobbold S, Hale G, Waldmann H: Non-lineage, LFA-1 family, and leucocyte common antigens: new and previously defined clusters. In: McMichael AJ (ed). Leucocyce Typing, White Cell Differentiation Antigens. New York, Oxford, 1987, pp 788-803
10.
Rudd CE, Morimoto C, Wong LL, Schlossman SF: The subdivision the T4 (CD4) subset on the basis of the differential expression L-C/T200 antigens. J Exp Med 166:17581773, 1987a
11.
Streuli M, Matsuyama T, Morimoto C, Schlossman SF, Saito H: Identification of the sequence required for expression of the 2H4 epitope on the human leukocyte common antigens. J Exp Med 166:15671572, 1987a
12.
Rudd CE, Morimoto C, Wong LL, Schlossman SF: The structure of the 2H4 antigen: a subset of the LCA/T200 family of antigens involved in immune suppression. In: McMichael AJ (ed). Leucocyte Typing, White Cell Differentiation Antigens. New York, Oxford, 1987, pp 242-245
13.
Ruoslahti E, Pierschbacher MD: New perspectives in cell adhesion: RGD and integrins. Science 238:491-497, 1987
14.
Sanders ME, Makgoba MW, Sharrow SO, Stephany D, Springer TA, Young HA, Shaw S: Human memory T lymphocytes express increased levels of three cell adhesion molecules (LFA-3, CD2, and LFA-1) and three other molecules (UCHLI, CDw29, and Pgp-1) and have enhanced IFN-gamma production. J 1mmuno1140:14011407, 1988a
15.
Ralfkiaer E, Lange Wantzin G: HTLV-positive and -negative cutaneous T-cell lymphomas in Denmark. An immunohistochemical study of T-cell antigens and T-cell subset associated antigens. In: McMichael AJ (ed). Leucocyte Typing, White Cell Differentiation Antigens. New York, Oxford, 1987, pp 290-291
16.
Sterry W: Mycosis fungoides. In: Berry CL (ed). Dermatopathology. Berlin, Springer, 1985, pp 167 - 223
17.
Suchi T, Lennert K, Tu LY, Kikuchi M, Sato E, Stansfeld AG, Feller AC: Histopathology and immunohistochemistry of peripheral Tcell lymphomas - a proposal for classification of T-cell lymphomas. Clin Path 40:995- 1015, 1987
18.
Stein H, Gerdes J, Schwab U, Lemke H, Mason DY, Ziegler A, Schienle W, Diehl V: Identification of Hodgkin and Sternberg Reed cells as a unique cell type derived from a newly detected small cell population. Int J Cancer 30:445 - 449, 1982
19.
Broder S, Edelson RL, Lutzner MA, Nelson DL, MacDermott RP, Durm ME, Goldman CK, Meade BD, Waldmann TA: The Sezary syndrome. A malignant proliferation of helper T cells. J Clin Invest 58:1297-1306, 1976
20.
Smith SH, Brown MH, Rowe D, Callard RE, Beverly PCL: Functional subsets of human helper-inducer cells defined by a new monoclonal antibody, UCHLI. Immunology 58:63-70, 1986
21.
De Paoli P, Battistin
the skin.
The excellent technical assistance of Miss S. Heimrich is gratefully acknowledged.
lymphocyte subsets: (suppressor inducer) 48:290 - 296, 1988
REFERENCES Romain PL, Schlossman Invest 74:1559-1565,
SF: Human 1984
T lymphocyte
subsets. J Clin
Morimoto C. Letvin NL, Discaso JA, Aldrich WR, Schlossman SF: The isolation and characterization of the human suppressor inducer T cell sub%et. J Immunol 134:1508-1514, 1985a Morimoto C, Letvin NL, Boyd AW, HaganM, Brown HM, Kornacki MM, Schlossman SF: The isolation and characterization of the
com-
of of
S, Santini GF: Age-related changes in human progressive reduction of the CD4 CD45R population. Clin Immunol Immunopathol
22.
Serra HM, Krowka JF, Ledbetter (Lp220) represents a post-thymic munol 140:1435-1441, 1988
23.
Chimenti S, Hoed1 S, Smolle J, Soyer HP, Torne R, Kerl H: Prognostic significance of plasma cell infiltrates in cutaneous T cell lymphomas. Am J Dermatopathol 9:167, 1987
JA, Pilarski LM: Loss of CD45R T cell differentiation event. J Im-