- Email: [email protected]
Locomotion of T cells from patients with cutaneous T-cell lymphoma (Sezary syndrome and mycosis fungoides)
CELLULAR
IMMUNOLOGY
50,
195-201(1980)
Locomotion of T Cells from Patients with Cutaneous T-Cell Lymphoma (Sezary Syndrome and Mycosis Fungoides) SUDHlR
GUPTA,
BIJAN
SAFAI,
RICHARD
EDELSON,*
ROBERT Memorial Sloan-Kettering Surgeons, Columbia
DELPHINE
PARRoTT,t
AND
GOOD
Cancer Center, New York, New York 10021, *College of Physicians University, New York, New York 10023, and TWestern Injrmary, University of Glasgow, Scotland Received
May
and
7, 1979
Peripheral blood T cells from eight patients with cutaneous lymphoma (four each with Sezary syndrome or mycosis fungoides) and T cells from skin tumor of one patient each with Sezary syndrome or mycosis fungoides were studied for their locomotor responses to the chemoattractant, casein. Nonmalignant peripheral blood T cells from each patient with mycosis fungoides moved normally. Malignant T cells from skin tumor of patients with mycosis fungoides or Sezary syndrome did not move in the presence of casein. Peripheral blood malignant T cells (Sezary cells) from three of four patients with Sezary syndrome either moved very poorly or did not move at all. The circulating Sezary cells from the fourth patient with Sezary syndrome responded moderately to the chemoattractant, casein. Two of three patients with Sezary syndrome with poor or no locomotor response of T cells underwent therapeutic leukopheresis without any demonstrable effect on their skin infiltration. The patient whose malignant T cells demonstrated moderate locomotor response to casein had a leukemic blast crisis and at that time her skin became free of malignant cells. A repeat study of her circulating T cells at that time demonstrated almost normal locomotor response to casein. These results demonstrate that the locomotor properties of malignant T cells in patients with Sezary syndrome may have prognostic significance.
INTRODUCTION The lymphoreticular neoplasms that are characterized by widespread infiltration of the skin have been regularly demonstrated to be T-cell malignancies, therefore, it has been suggested that they be grouped together under the term cutaneous T-cell lymphoma (1). The leukemic Sezary syndrome and the aleukemic mycosis fungoides are a part of this spectrum. Sezary syndrome is a disorder characterized by generalized exofoliative erythroderma, intensive pruritis, and the presence of atypical “malignant” cells in the peripheral blood as well as in the cellular infiltrate of the skin. Mycosis fungoides is clinically characterized by erythematous patches, infiltrated plaques, and tumors which progress usually over a period of years to involve internal organs. Malignant cells are confined to the skin and are usually lacking in the peripheral blood of patients with mycosis fungoides. Both mycosis fungoides atypical cells (MF cells) and Sezary syndrome cells (SS cells) have common features. Both have characteristic folded, cerebriform nucleus, and scanty cytoplasm and they bind to native sheep erythrocytes (SRBC). The latter 195 000%8749/80/030195-07$02.00/O Copyright All
rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
196
SHORT
COMMUNICATIONS
characteristic identified them as cells of T-cell lineage (2-9). Human T lymphocytes have been subdivided into two distinct subpopulations as identified by the presence of receptors for IgM (TE.L cells) or IgG (Tr cells) (10, 11). We have recently demonstrated that Tp cells move well in vitro toward the chemoattractant, casein, and Tr cells either do not move or move very poorly toward casein (12). Furthermore, we have also shown that when T cells are separated according to surface charge density gradient electrophoresis, Tp cells are found in high mobility fractions and Tr in low mobility fractions (13). Edelson et al. (14) have reported a beneficial effect of leukopheresis on skin infiltrations in patients with Sezary syndrome. The present study was done to investigate (a) the differences in locomotor properties of malignant T cells in the peripheral blood from patients with Sezary syndrome and nonmalignant peripheral blood T cells in patients with mycosis fungoides, (b) whether there is any correlation between locomotion and clinical course of disease particularly with regard to skin infiltration, and (c) whether the locomotion of T cells correlates with the surface phenotypes, e.g., the expression of receptors for IgM (Tp) or IgG (Ty). MATERIAL
AND METHODS
Material
Four patients with mycosis fungoides and five patients with Sezary syndrome attending Dermatology Clinics of the Memorial Hospital and Columbia Presbyterian Medical Center were the subjects of the present study. Diagnosis was made by clinical, histological, and cytological examination. Peripheral blood and skin tumor cells from patients with Sezary syndrome and skin tumor cells from a patient with mycosis fungoides had 90-98% cells with the typical morphological characteristics of SS or MF cells as examined by both light and electron microscopy. More than 90% of these atypical cells formed spontaneous rosettes with SRBC. Peripheral blood lymphocytes from patients with mycosis fungoides had only occasional MF cells. Fifteen healthy age and sex-matched subjects served as normal controls. Methods Isolation of mononuclear cells. Peripheral heparinized venous blood mononuclear cells were isolated on Ficoll-Hypaque (FH) gradient. Cells from skin tumors were teased apart and passed through wire mesh to obtain a single cell suspension. Cells were washed three times in Hanks’ balanced salt solution (HBSS) and resuspended in RPMI-1640 (Grand Island Biological Company, Grand Island, N.Y .) containing, 20% fetal calf serum (FCS) at a concentration of 4 x lO’Vm1. To the cell suspension, in a ratio of 2: 1, was added carbonyl iron (Lymphocytes Separator Reagent, Technicon, Tarrytown, N.Y.). The mixture was incubated on a rotator at 37°C for 30 min, and phagocytic cells were separated from nonphagocytic lymphoid cells on FH gradient. Lymphoid cells contained l-2% peroxidasepositive cells and viability was approximately 98% as determined by trypan blue dye exclusion test. Purification of T cells. One-millimeter aliquots of lymphoid cells depleted of phagocytic cells (4 x lo6 cells/ml) were mixed with 1 ml of neuraminidase-treated sheep erythrocytes (SRBC) and 0.25 ml of heat-inactivated fetal calf serum
SHORT
COMMUNICATIONS
197
(absorbed with SRBC). The mixture was incubated at 37°C for 5 min, centrifuged for 5 min at 200 g and incubated at 4°C for 1 hr. The pellets were resuspended and layered over FH and centrifuged for 20 min at 4OOg. T cells rosetted with SRBC (pellet) were separated from non-T cells at the interface. Erythrocytes attached to T cells were lysed with Tris buffer containing 0.83% ammonium chloride (pH 7.2). T cells were washed in RPMI-1640 three times and resuspended in RPMI-1640 containing 20% FCS at a concentration of 4 x 10Vml. The purity of T cells was approximately 98%. T-Cell suspensions were incubated overnight at 37°C in a humidified atmosphere of 5% CO, and 95% air. Following incubation cells were washed three times in Gey’s balanced salt solution (Grand Island Biological Company, Grand Island, N.Y.). A small sample of T-cell suspension was used for the determination of Tp- and Ty-cell proportions. For the chemotactic assay, cells were resuspended in Gey’s solution at a concentration of 2 x lO”/ml. Locomotion assay. Casein (Merck, Darmstadt, West Germany), at a concentration of 1 mg/ml, was used to promote locomotion. Gey’s solution was used as the negative control. The tests were performed in modified Boyden chambers as described by Wilkinson (15). In all experiments, filters of 8-m pore size (Milipore Corp., Bedford, Mass.) were used and incubated for 3 hr at 37°C in a humidified atmosphere at 5% CO, and 95% air. Cell locomotion was assayed by the leading front method (16) which measures the distance, micrometers, that cells migrate from the upper compartment through micropore filters, in response to a chemoattractant placed in the lower compartment. TF and Ty cells were assayed by the method described (17) and results are presented as percentage of T cells. RESULTS The results of locomotion of T cells toward the chemoattractant, casein, are shown in Table 1. Peripheral blood T cells from patients 1,2, and 3 and T cells from skin tumor of patient 5, most of which were Sezary cells, did not move or moved very poorly in the presence of casein. Peripheral blood T cells (Sezary cells) from patient 4 (McB) responded moderately to the chemotactic stimulus of casein. This patient after 3 months had a leukemic blastic crisis and at that time her skin became free of Sezary cells (proved by skin biopsy on three occasions). A repeat study demonstrated a normal locomotor response to casein. Peripheral blood T cells from all four patients demonstrated locomotor response to casein comparable to that of T cells from normal controls. Mycosis fungoides T cells from the skin tumor of a patient (Aa) migrated very poorly toward the chemoattractant, casein. No correlation was observed between the proportions of Tp and Ty cells and locomotion of T cells in the patients with mycosis fungoids or Sezary syndrome. Data regarding T cell subsets are shown in Table 2. DISCUSSION Both Sezary syndrome and mycosis fungoides, a variety of cutaneous T-cell lymphoma, represent disorders characterized by skin infiltration with atypical cells characterized by folded and cerebriform nuclei and scanty cytoplasm. MF cells and SS cells form spontaneous rosettes with SRBC, a property considered to be a characteristic of human T lymphocytes. MF cells are confined to the skin and
198
SHORT COMMUNICATIONS TABLE Locomotion
1
of Malignant T Cells from Patients with Sezary Syndrome and Nonmalignant T Cells from Mycosis Fungoides Distance of migration in pm (mean + SD)
No.
Patient
Sezary syndrome 1 2 3 4
GP SN SE McB
5
OD
Mycosis fungoides 1 2 3
RI EN Aa
4
Ro
Source of cllls
Geys
PBL PBL PBL PBL PBL” Skin tumor
9+2 3*4 16 + 10 6+2 10 + 2 s+3
PBL PBL PBL Skin tumor PBL
8+3 29 + 12 + 15 + 12 +
Controls
PBL
Casein
5 4 3 5
12 + 10
24 9 24 46 70 19
+ + 2 + + +
7 13 15 10 12 5
71 + 114 + 105 + 26 + 110 +
19 12 16 2 14
90 + 15
(’ Repeat study at a time when patient’s skin became clear of malignant cells. PBL = peripheral blood.
generally are lacking from the peripheral blood. By contrast, in patients with Sezary syndrome, SS cells are present both in the skin and in their circulation. Atypical SS cells have recently been reported to be heterogeneous with regard to the expression of receptor for IgM or IgG (9). In the present study we have also observed a similar TABLE
2
T Cell Subsets in Patients with Sezary Syndrome and Mycosis Fungoides SN Sezary syndrome 1 2 3 4 5 Mycosis fungoides 1 2 3 4 Controls
Patient GP SN SE McB OD RI EN Aa Ro
Source of cells
Malignant cell (%I
TP (%I
PBL PBL PBL PBL PBL” Skin tumor
95.0 94.0 90.0 97.0 98.0 99.9
50.0 10.0 50.0 1.0 1.0 0.0
0.0 0.0 0.0 1.0 1.0 0.0
PBL PBL PBL Skin tumor PBL
-
29.0 37.0 40.0 51.0 51.0
16.0 10.0 10.0 10.0 10.0
52 -t 10.2
10.4 k 2.8
PBL
95.0 -
D Repeat study at a time when patient’s skin became clear of malignant cells.
TY (%)
SHORT
COMMUNICATIONS
199
heterogeneity of SS cells and MF cells. Edelson et al. (14) have proposed the view that in the Sezary syndrome, SS cells are in a state of equilibrium between skin and the circulating blood and, therefore, when the peripheral blood pool of lymphocytes (majority of which are SS cells) is depleted by leukopheresis, mobilization of cells from the skin with clinical improvement of skin lesions occurs. Furthermore, extension of the clinical trial of leukopheresis lead the same group to suggest that response to this therapeutic modality may correlate with the rate of cell renewal of the neoplastic cells (18). In that study it was demonstrated that three individuals who responded to leukopheresis had circulating neoplastic T cells which spontaneously incorporated little tritiated thymidine. Those patients with rapid spontaneous DNA synthesis by their circulating neoplastic cells responded poorly, suggesting that at least one factor governing the efficacy of leukopheresis in these patients is the ability to remove neoplastic cells far more rapidly than they are being produced. The present study suggests that an additional factor may involve locomotor properties of the neoplastic cells. It will be important to prospectively study individual patients while they are undergoing therapy to determine whether this particular feature of the neoplastic cells is of prognostic significance. Safai et al. (19) have also reported the beneficial effect of leukopheresis on skin lesions is only in a subgroup of patients with Sezary syndrome. Therefore, it is possible that it is the intrinsic property of locomotion of the malignant cells which determines the success of leukopheresis treatment. Lymphocytes and lymphoblasts from humans will show locomotor activity in response to various chemoattractants (20, 21). Recently we have reported that Tp cells containing a subpopulation of cells with helper activity for the differentiation of B cells to plasma cells move very well toward chemoattractant, casein, and Ty cells containing a subpopulation of cells with suppressor activity do not move toward casein (12). Recently we have examined the locomotion of T cells and T-cell subsets to determine whether the locomotion is directed (chemotaxis) or nondirected (chemokinesis) (22). T cells, Tp and Ty cells, all exhibited chemokinesis rather than chemotaxis when casein was used as a chemoattractant. Therefore, the locomotion of T cells observed in the present study of patients with Sezary syndrome and mycosis fungoides appears to be chemokinesis. McCarty and Goetzl(23) also reported chemokinesis of human T cells that could be stimulated by arachidonic acid. Broder and associates (24) have recently demonstrated that malignant Sezary cells represent a monoclonal expansion of helper T cells, which when put in cocultures with normal B cells in presence of pokeweed mitogen, enhanced immunoglobulin production by the B lymphocytes. In the present study, however, no such correlation between helper activity (data not shown), receptors for IgM (T/J cells), and locomotor response to casein was observed. Sezary T cells both with or without IgM receptors had poor locomotor response. Similarly MF cells from skin tumor of one patient had no receptors for either IgM or IgG and did not move in the presence of casein. Thymocytes in general move very poorly in the presence of casein and lack receptors for both IgM and IgG ((25) and unpublished data). It appears likely that the atypical malignant cells in the skin of Sezary syndrome or mycosis fungoides and in the peripheral blood of patients with Sezary syndrome have functional and phenotypic characteristics of thymocytes but the cells are more differentiated than those in the thymus but less mature than normal circulating T cells. Nonmalignant T cells in the peripheral blood of patients with
200
SHORT
COMMUNICATIONS
mycosis fungoides had normal locomotor response to casein. However, malignant T cells from the skin tumor of either patients with mycosis fungoides or Sezary syndrome had a very poor locomotor activity in the presence of the chemoattractant. Malignant T cells from patient McB moved to a moderate distance into the filter in the presence of casein. Three months following this initial study, the peripheral blood counts increased from 22,50O/mm” to 200,00O/cm” most of which represented a monoclonal expansion of SS cells by cytogenetic and electron microscopic examination. At this time her skin which had shown malignant cells on biopsy became free of the malignant cells. A repeat study of peripheral blood SS cells at this time demonstrated no alteration in the surface phenotype for IgM or IgG but the locomotor response of the cells was further increased and reached the range that features normal T cells. Two patients (GP and SE) were subjected to leukophoresis but the latter procedure had no effect on the density of skin infiltration with the malignant cells. From these studies of cell locomotion and effect of leukophoresis and blastic crisis in this disease on skin infiltrates, it seems likely that the malignant T cells with moderate to good locomotor response to chemoattractants could respond to the treatment with leukophoresis while those with poor or no locomotor activity of malignant cells might be resistant to this therapeutic effort. A prospective study to explore this view is now in progress. Abnormalities of T-cell locomotor response have also been observed in the peripheral blood and spleen from untreated patients with Hodgkin’s disease (26). These abnormalities of locomotion have been observed in association with abnormal distribution of the T-cell subsets between the peripheral blood and spleen from patients with Hodgkin’s disease (26). What makes T cells go to the skin in mycosis fungoides or Sezary syndrome remains an unanswered question. There is a possibility that a chemotactic factor is generated locally in the skin perhaps as a result of in inflammatory process which can act on malignant cells and influences or determines the accumulation of these cells. But if this be the case it seems likely that in some cases, at least, the cells develop or change as a consequence of residence in the skin and the change interferes with their capacity to respond to locomotor stimuli. ACKNOWLEDGMENTS We wish to acknowledge the expert technical assistance of Ms. S. Khanna, Ms. S. Garber, Ms. E. Frederick, and Mr. A. Prajapathi. This work was supported by grants from National Institutes of Health CA-17404, CA-19267, CA-08748, CA-20499, AI-11843, NS-11457, the Fund for the Advanced Study of Cancer, Witty Fund, and Chesebrough Ponds Inc.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Edelson, R. L., Ann. Int. Med. 83, 536, 1975. Rosas-Uribe, A., Variakojis, D., Molnarz, R., and Rappaport, H., Cancer 34, 634, 1974. Lutzner, M. A., Edelson, R. L., Smith, R. W., Shevach, E. M., and Green, I., Lancer 2,207,1973. Brouet, J. C., Flandrin, G., and Seligmann, M., N. Engl. J. Med. 29, 293, 1973. Broome, J. D., Zucker-Franklin. D., Weiner, M. S., Bianco, C., and Nussenzweig, V., C/in. Immunol. Immunopathol. 1, 315, 1973. Robinowitz, B. N., Noguchi, S., and Roenigk, H. H., Cancer 37, 1747, 1978. Gupta, S., Safai, B., and Good, R. A., Amer. J. Hematol. 4, 133, 1978. Gupta, S., Good, R. A., and Siegal, F. P., Clin. Exp. Immunol. 26, 204, 1976. Gupta, S., and Good, R. A., Cell. Immunol. 39, 18, 1978. Ferrarini, M., Moretta, L., and Abrile, R., et al., Eur. J. Immunol. 5, 70, 1975.
SHORT
COMMUNICATIONS
201
I I, Moretta, L., Ferrarini, M., Durante, M. L., and Mingari, M. C., Eur. .I. fmmunol. 5, 565, 1975. 12. Parrott, D. M. V., Good, R. A., O’Neill, G. J., and Gupta, S., Proc. Nat. Acad. Sci. USA 75,2392, 1978. 13. Platsoucas, C. D., Good, R. A., and Gupta, S., Proc. Nut. Acad. Sci. USA 76, 1972, 1979. 14. Edelson, R., Facktor, M., Andrews, A., Lutzner, M., and Schein, P., N. EngI. .I. Med. 291, 293, 1974. 15. Wilkinson, P. C., “Chemotaxis and Inflammation, p. 33. Churchill-Livingston, Edinburgh, Scotland, 1973. 16. Sigmond. S. H., and Hirsch. J. G., J. &p. Med. 137, 387, 1973. 17. Gupta, S., and Good, R. A., Cell. Immunol. 34, 10, 1977. 18. Edelson, R. L., “Proceedings of the 4th Advanced Blood Components Seminar,” p. I, 1977. 19. Safai, B., Reich, L., and Good. R. A., Chin. Res. 26, 576A. 1978. 20. Russell, R. J., Wilkinson, P. C., Sless, F., and Parrott, D. M. V. Nature (London) 256, 646, 1975. 21. O’Neill, G. J., and Parrott, D. M. V., Cell. Immrtnol. 33, 257, 1977. 22. Gupta, S., and Good, R. A., Thymrrs 1, 135, 1979. 23. McCarty. J., and Goetzl, E. J., Cell. Immune/. 43, 103, 1979. 24. Broder, S., Edelson, R. L., Lutzner, M. S., Nelson, D. L., MacDermott, R. P., Durm, M., Goldman, E., Meade, C. K., and Waldmann, T. A., J. C/in. Invest. 58, 1297, 1976. 25. Gupta. S., and Good, R. A., Cell. Immunol. 36, 263, 1978. 26. Gupta. S.. Tan, C., and Good, R. A.. Fed. Proc. 38, 1162, 1979.
IMMUNOLOGY
50,
195-201(1980)
Locomotion of T Cells from Patients with Cutaneous T-Cell Lymphoma (Sezary Syndrome and Mycosis Fungoides) SUDHlR
GUPTA,
BIJAN
SAFAI,
RICHARD
EDELSON,*
ROBERT Memorial Sloan-Kettering Surgeons, Columbia
DELPHINE
PARRoTT,t
AND
GOOD
Cancer Center, New York, New York 10021, *College of Physicians University, New York, New York 10023, and TWestern Injrmary, University of Glasgow, Scotland Received
May
and
7, 1979
Peripheral blood T cells from eight patients with cutaneous lymphoma (four each with Sezary syndrome or mycosis fungoides) and T cells from skin tumor of one patient each with Sezary syndrome or mycosis fungoides were studied for their locomotor responses to the chemoattractant, casein. Nonmalignant peripheral blood T cells from each patient with mycosis fungoides moved normally. Malignant T cells from skin tumor of patients with mycosis fungoides or Sezary syndrome did not move in the presence of casein. Peripheral blood malignant T cells (Sezary cells) from three of four patients with Sezary syndrome either moved very poorly or did not move at all. The circulating Sezary cells from the fourth patient with Sezary syndrome responded moderately to the chemoattractant, casein. Two of three patients with Sezary syndrome with poor or no locomotor response of T cells underwent therapeutic leukopheresis without any demonstrable effect on their skin infiltration. The patient whose malignant T cells demonstrated moderate locomotor response to casein had a leukemic blast crisis and at that time her skin became free of malignant cells. A repeat study of her circulating T cells at that time demonstrated almost normal locomotor response to casein. These results demonstrate that the locomotor properties of malignant T cells in patients with Sezary syndrome may have prognostic significance.
INTRODUCTION The lymphoreticular neoplasms that are characterized by widespread infiltration of the skin have been regularly demonstrated to be T-cell malignancies, therefore, it has been suggested that they be grouped together under the term cutaneous T-cell lymphoma (1). The leukemic Sezary syndrome and the aleukemic mycosis fungoides are a part of this spectrum. Sezary syndrome is a disorder characterized by generalized exofoliative erythroderma, intensive pruritis, and the presence of atypical “malignant” cells in the peripheral blood as well as in the cellular infiltrate of the skin. Mycosis fungoides is clinically characterized by erythematous patches, infiltrated plaques, and tumors which progress usually over a period of years to involve internal organs. Malignant cells are confined to the skin and are usually lacking in the peripheral blood of patients with mycosis fungoides. Both mycosis fungoides atypical cells (MF cells) and Sezary syndrome cells (SS cells) have common features. Both have characteristic folded, cerebriform nucleus, and scanty cytoplasm and they bind to native sheep erythrocytes (SRBC). The latter 195 000%8749/80/030195-07$02.00/O Copyright All
rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
196
SHORT
COMMUNICATIONS
characteristic identified them as cells of T-cell lineage (2-9). Human T lymphocytes have been subdivided into two distinct subpopulations as identified by the presence of receptors for IgM (TE.L cells) or IgG (Tr cells) (10, 11). We have recently demonstrated that Tp cells move well in vitro toward the chemoattractant, casein, and Tr cells either do not move or move very poorly toward casein (12). Furthermore, we have also shown that when T cells are separated according to surface charge density gradient electrophoresis, Tp cells are found in high mobility fractions and Tr in low mobility fractions (13). Edelson et al. (14) have reported a beneficial effect of leukopheresis on skin infiltrations in patients with Sezary syndrome. The present study was done to investigate (a) the differences in locomotor properties of malignant T cells in the peripheral blood from patients with Sezary syndrome and nonmalignant peripheral blood T cells in patients with mycosis fungoides, (b) whether there is any correlation between locomotion and clinical course of disease particularly with regard to skin infiltration, and (c) whether the locomotion of T cells correlates with the surface phenotypes, e.g., the expression of receptors for IgM (Tp) or IgG (Ty). MATERIAL
AND METHODS
Material
Four patients with mycosis fungoides and five patients with Sezary syndrome attending Dermatology Clinics of the Memorial Hospital and Columbia Presbyterian Medical Center were the subjects of the present study. Diagnosis was made by clinical, histological, and cytological examination. Peripheral blood and skin tumor cells from patients with Sezary syndrome and skin tumor cells from a patient with mycosis fungoides had 90-98% cells with the typical morphological characteristics of SS or MF cells as examined by both light and electron microscopy. More than 90% of these atypical cells formed spontaneous rosettes with SRBC. Peripheral blood lymphocytes from patients with mycosis fungoides had only occasional MF cells. Fifteen healthy age and sex-matched subjects served as normal controls. Methods Isolation of mononuclear cells. Peripheral heparinized venous blood mononuclear cells were isolated on Ficoll-Hypaque (FH) gradient. Cells from skin tumors were teased apart and passed through wire mesh to obtain a single cell suspension. Cells were washed three times in Hanks’ balanced salt solution (HBSS) and resuspended in RPMI-1640 (Grand Island Biological Company, Grand Island, N.Y .) containing, 20% fetal calf serum (FCS) at a concentration of 4 x lO’Vm1. To the cell suspension, in a ratio of 2: 1, was added carbonyl iron (Lymphocytes Separator Reagent, Technicon, Tarrytown, N.Y.). The mixture was incubated on a rotator at 37°C for 30 min, and phagocytic cells were separated from nonphagocytic lymphoid cells on FH gradient. Lymphoid cells contained l-2% peroxidasepositive cells and viability was approximately 98% as determined by trypan blue dye exclusion test. Purification of T cells. One-millimeter aliquots of lymphoid cells depleted of phagocytic cells (4 x lo6 cells/ml) were mixed with 1 ml of neuraminidase-treated sheep erythrocytes (SRBC) and 0.25 ml of heat-inactivated fetal calf serum
SHORT
COMMUNICATIONS
197
(absorbed with SRBC). The mixture was incubated at 37°C for 5 min, centrifuged for 5 min at 200 g and incubated at 4°C for 1 hr. The pellets were resuspended and layered over FH and centrifuged for 20 min at 4OOg. T cells rosetted with SRBC (pellet) were separated from non-T cells at the interface. Erythrocytes attached to T cells were lysed with Tris buffer containing 0.83% ammonium chloride (pH 7.2). T cells were washed in RPMI-1640 three times and resuspended in RPMI-1640 containing 20% FCS at a concentration of 4 x 10Vml. The purity of T cells was approximately 98%. T-Cell suspensions were incubated overnight at 37°C in a humidified atmosphere of 5% CO, and 95% air. Following incubation cells were washed three times in Gey’s balanced salt solution (Grand Island Biological Company, Grand Island, N.Y.). A small sample of T-cell suspension was used for the determination of Tp- and Ty-cell proportions. For the chemotactic assay, cells were resuspended in Gey’s solution at a concentration of 2 x lO”/ml. Locomotion assay. Casein (Merck, Darmstadt, West Germany), at a concentration of 1 mg/ml, was used to promote locomotion. Gey’s solution was used as the negative control. The tests were performed in modified Boyden chambers as described by Wilkinson (15). In all experiments, filters of 8-m pore size (Milipore Corp., Bedford, Mass.) were used and incubated for 3 hr at 37°C in a humidified atmosphere at 5% CO, and 95% air. Cell locomotion was assayed by the leading front method (16) which measures the distance, micrometers, that cells migrate from the upper compartment through micropore filters, in response to a chemoattractant placed in the lower compartment. TF and Ty cells were assayed by the method described (17) and results are presented as percentage of T cells. RESULTS The results of locomotion of T cells toward the chemoattractant, casein, are shown in Table 1. Peripheral blood T cells from patients 1,2, and 3 and T cells from skin tumor of patient 5, most of which were Sezary cells, did not move or moved very poorly in the presence of casein. Peripheral blood T cells (Sezary cells) from patient 4 (McB) responded moderately to the chemotactic stimulus of casein. This patient after 3 months had a leukemic blastic crisis and at that time her skin became free of Sezary cells (proved by skin biopsy on three occasions). A repeat study demonstrated a normal locomotor response to casein. Peripheral blood T cells from all four patients demonstrated locomotor response to casein comparable to that of T cells from normal controls. Mycosis fungoides T cells from the skin tumor of a patient (Aa) migrated very poorly toward the chemoattractant, casein. No correlation was observed between the proportions of Tp and Ty cells and locomotion of T cells in the patients with mycosis fungoids or Sezary syndrome. Data regarding T cell subsets are shown in Table 2. DISCUSSION Both Sezary syndrome and mycosis fungoides, a variety of cutaneous T-cell lymphoma, represent disorders characterized by skin infiltration with atypical cells characterized by folded and cerebriform nuclei and scanty cytoplasm. MF cells and SS cells form spontaneous rosettes with SRBC, a property considered to be a characteristic of human T lymphocytes. MF cells are confined to the skin and
198
SHORT COMMUNICATIONS TABLE Locomotion
1
of Malignant T Cells from Patients with Sezary Syndrome and Nonmalignant T Cells from Mycosis Fungoides Distance of migration in pm (mean + SD)
No.
Patient
Sezary syndrome 1 2 3 4
GP SN SE McB
5
OD
Mycosis fungoides 1 2 3
RI EN Aa
4
Ro
Source of cllls
Geys
PBL PBL PBL PBL PBL” Skin tumor
9+2 3*4 16 + 10 6+2 10 + 2 s+3
PBL PBL PBL Skin tumor PBL
8+3 29 + 12 + 15 + 12 +
Controls
PBL
Casein
5 4 3 5
12 + 10
24 9 24 46 70 19
+ + 2 + + +
7 13 15 10 12 5
71 + 114 + 105 + 26 + 110 +
19 12 16 2 14
90 + 15
(’ Repeat study at a time when patient’s skin became clear of malignant cells. PBL = peripheral blood.
generally are lacking from the peripheral blood. By contrast, in patients with Sezary syndrome, SS cells are present both in the skin and in their circulation. Atypical SS cells have recently been reported to be heterogeneous with regard to the expression of receptor for IgM or IgG (9). In the present study we have also observed a similar TABLE
2
T Cell Subsets in Patients with Sezary Syndrome and Mycosis Fungoides SN Sezary syndrome 1 2 3 4 5 Mycosis fungoides 1 2 3 4 Controls
Patient GP SN SE McB OD RI EN Aa Ro
Source of cells
Malignant cell (%I
TP (%I
PBL PBL PBL PBL PBL” Skin tumor
95.0 94.0 90.0 97.0 98.0 99.9
50.0 10.0 50.0 1.0 1.0 0.0
0.0 0.0 0.0 1.0 1.0 0.0
PBL PBL PBL Skin tumor PBL
-
29.0 37.0 40.0 51.0 51.0
16.0 10.0 10.0 10.0 10.0
52 -t 10.2
10.4 k 2.8
PBL
95.0 -
D Repeat study at a time when patient’s skin became clear of malignant cells.
TY (%)
SHORT
COMMUNICATIONS
199
heterogeneity of SS cells and MF cells. Edelson et al. (14) have proposed the view that in the Sezary syndrome, SS cells are in a state of equilibrium between skin and the circulating blood and, therefore, when the peripheral blood pool of lymphocytes (majority of which are SS cells) is depleted by leukopheresis, mobilization of cells from the skin with clinical improvement of skin lesions occurs. Furthermore, extension of the clinical trial of leukopheresis lead the same group to suggest that response to this therapeutic modality may correlate with the rate of cell renewal of the neoplastic cells (18). In that study it was demonstrated that three individuals who responded to leukopheresis had circulating neoplastic T cells which spontaneously incorporated little tritiated thymidine. Those patients with rapid spontaneous DNA synthesis by their circulating neoplastic cells responded poorly, suggesting that at least one factor governing the efficacy of leukopheresis in these patients is the ability to remove neoplastic cells far more rapidly than they are being produced. The present study suggests that an additional factor may involve locomotor properties of the neoplastic cells. It will be important to prospectively study individual patients while they are undergoing therapy to determine whether this particular feature of the neoplastic cells is of prognostic significance. Safai et al. (19) have also reported the beneficial effect of leukopheresis on skin lesions is only in a subgroup of patients with Sezary syndrome. Therefore, it is possible that it is the intrinsic property of locomotion of the malignant cells which determines the success of leukopheresis treatment. Lymphocytes and lymphoblasts from humans will show locomotor activity in response to various chemoattractants (20, 21). Recently we have reported that Tp cells containing a subpopulation of cells with helper activity for the differentiation of B cells to plasma cells move very well toward chemoattractant, casein, and Ty cells containing a subpopulation of cells with suppressor activity do not move toward casein (12). Recently we have examined the locomotion of T cells and T-cell subsets to determine whether the locomotion is directed (chemotaxis) or nondirected (chemokinesis) (22). T cells, Tp and Ty cells, all exhibited chemokinesis rather than chemotaxis when casein was used as a chemoattractant. Therefore, the locomotion of T cells observed in the present study of patients with Sezary syndrome and mycosis fungoides appears to be chemokinesis. McCarty and Goetzl(23) also reported chemokinesis of human T cells that could be stimulated by arachidonic acid. Broder and associates (24) have recently demonstrated that malignant Sezary cells represent a monoclonal expansion of helper T cells, which when put in cocultures with normal B cells in presence of pokeweed mitogen, enhanced immunoglobulin production by the B lymphocytes. In the present study, however, no such correlation between helper activity (data not shown), receptors for IgM (T/J cells), and locomotor response to casein was observed. Sezary T cells both with or without IgM receptors had poor locomotor response. Similarly MF cells from skin tumor of one patient had no receptors for either IgM or IgG and did not move in the presence of casein. Thymocytes in general move very poorly in the presence of casein and lack receptors for both IgM and IgG ((25) and unpublished data). It appears likely that the atypical malignant cells in the skin of Sezary syndrome or mycosis fungoides and in the peripheral blood of patients with Sezary syndrome have functional and phenotypic characteristics of thymocytes but the cells are more differentiated than those in the thymus but less mature than normal circulating T cells. Nonmalignant T cells in the peripheral blood of patients with
200
SHORT
COMMUNICATIONS
mycosis fungoides had normal locomotor response to casein. However, malignant T cells from the skin tumor of either patients with mycosis fungoides or Sezary syndrome had a very poor locomotor activity in the presence of the chemoattractant. Malignant T cells from patient McB moved to a moderate distance into the filter in the presence of casein. Three months following this initial study, the peripheral blood counts increased from 22,50O/mm” to 200,00O/cm” most of which represented a monoclonal expansion of SS cells by cytogenetic and electron microscopic examination. At this time her skin which had shown malignant cells on biopsy became free of the malignant cells. A repeat study of peripheral blood SS cells at this time demonstrated no alteration in the surface phenotype for IgM or IgG but the locomotor response of the cells was further increased and reached the range that features normal T cells. Two patients (GP and SE) were subjected to leukophoresis but the latter procedure had no effect on the density of skin infiltration with the malignant cells. From these studies of cell locomotion and effect of leukophoresis and blastic crisis in this disease on skin infiltrates, it seems likely that the malignant T cells with moderate to good locomotor response to chemoattractants could respond to the treatment with leukophoresis while those with poor or no locomotor activity of malignant cells might be resistant to this therapeutic effort. A prospective study to explore this view is now in progress. Abnormalities of T-cell locomotor response have also been observed in the peripheral blood and spleen from untreated patients with Hodgkin’s disease (26). These abnormalities of locomotion have been observed in association with abnormal distribution of the T-cell subsets between the peripheral blood and spleen from patients with Hodgkin’s disease (26). What makes T cells go to the skin in mycosis fungoides or Sezary syndrome remains an unanswered question. There is a possibility that a chemotactic factor is generated locally in the skin perhaps as a result of in inflammatory process which can act on malignant cells and influences or determines the accumulation of these cells. But if this be the case it seems likely that in some cases, at least, the cells develop or change as a consequence of residence in the skin and the change interferes with their capacity to respond to locomotor stimuli. ACKNOWLEDGMENTS We wish to acknowledge the expert technical assistance of Ms. S. Khanna, Ms. S. Garber, Ms. E. Frederick, and Mr. A. Prajapathi. This work was supported by grants from National Institutes of Health CA-17404, CA-19267, CA-08748, CA-20499, AI-11843, NS-11457, the Fund for the Advanced Study of Cancer, Witty Fund, and Chesebrough Ponds Inc.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Edelson, R. L., Ann. Int. Med. 83, 536, 1975. Rosas-Uribe, A., Variakojis, D., Molnarz, R., and Rappaport, H., Cancer 34, 634, 1974. Lutzner, M. A., Edelson, R. L., Smith, R. W., Shevach, E. M., and Green, I., Lancer 2,207,1973. Brouet, J. C., Flandrin, G., and Seligmann, M., N. Engl. J. Med. 29, 293, 1973. Broome, J. D., Zucker-Franklin. D., Weiner, M. S., Bianco, C., and Nussenzweig, V., C/in. Immunol. Immunopathol. 1, 315, 1973. Robinowitz, B. N., Noguchi, S., and Roenigk, H. H., Cancer 37, 1747, 1978. Gupta, S., Safai, B., and Good, R. A., Amer. J. Hematol. 4, 133, 1978. Gupta, S., Good, R. A., and Siegal, F. P., Clin. Exp. Immunol. 26, 204, 1976. Gupta, S., and Good, R. A., Cell. Immunol. 39, 18, 1978. Ferrarini, M., Moretta, L., and Abrile, R., et al., Eur. J. Immunol. 5, 70, 1975.
SHORT
COMMUNICATIONS
201
I I, Moretta, L., Ferrarini, M., Durante, M. L., and Mingari, M. C., Eur. .I. fmmunol. 5, 565, 1975. 12. Parrott, D. M. V., Good, R. A., O’Neill, G. J., and Gupta, S., Proc. Nat. Acad. Sci. USA 75,2392, 1978. 13. Platsoucas, C. D., Good, R. A., and Gupta, S., Proc. Nut. Acad. Sci. USA 76, 1972, 1979. 14. Edelson, R., Facktor, M., Andrews, A., Lutzner, M., and Schein, P., N. EngI. .I. Med. 291, 293, 1974. 15. Wilkinson, P. C., “Chemotaxis and Inflammation, p. 33. Churchill-Livingston, Edinburgh, Scotland, 1973. 16. Sigmond. S. H., and Hirsch. J. G., J. &p. Med. 137, 387, 1973. 17. Gupta, S., and Good, R. A., Cell. Immunol. 34, 10, 1977. 18. Edelson, R. L., “Proceedings of the 4th Advanced Blood Components Seminar,” p. I, 1977. 19. Safai, B., Reich, L., and Good. R. A., Chin. Res. 26, 576A. 1978. 20. Russell, R. J., Wilkinson, P. C., Sless, F., and Parrott, D. M. V. Nature (London) 256, 646, 1975. 21. O’Neill, G. J., and Parrott, D. M. V., Cell. Immrtnol. 33, 257, 1977. 22. Gupta, S., and Good, R. A., Thymrrs 1, 135, 1979. 23. McCarty. J., and Goetzl, E. J., Cell. Immune/. 43, 103, 1979. 24. Broder, S., Edelson, R. L., Lutzner, M. S., Nelson, D. L., MacDermott, R. P., Durm, M., Goldman, E., Meade, C. K., and Waldmann, T. A., J. C/in. Invest. 58, 1297, 1976. 25. Gupta. S., and Good, R. A., Cell. Immunol. 36, 263, 1978. 26. Gupta. S.. Tan, C., and Good, R. A.. Fed. Proc. 38, 1162, 1979.