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Subpopulations of human T lymphocytes
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Subpopulations
15,
133-143 (1980)
of Human
T Lymphocytes
XIV. Abnormality of T-Cell Locomotion and of Distribution of Subpopulations of T and 6 Lymphocytes in Peripheral Blood and Spleen from Children with Untreated Hodgkin’s Disease SUDHIR Memoriul
Sloan-Kettering
GUPTA Cancer
AND CHARLOTTE Center.
New
York.
TAN New,
York
10021
Received June 13. 1979 Peripheral blood and splenic mononuclear cells from untreated children with Hodgkin’s disease were examined for the proportions of T, B, and third population cells, T cells with IgM(Tp) or IgG(Ty1 receptors, locomotion of T cells toward chemoattractants, casein- and endotoxin-activated serum, and for the effect of splenectomy on T-cell subsets and on locomotion of T cells. In the peripheral blood, proportions of T, B, and third population cells were comparable to those of controls, however, spleen contained increased proportions of T cells and decreased proportion of B lymphocytes. TF cells were comparable and Ty cells were moderately increased in the peripheral blood resulting in an abnormally low ratio of T&/Ty cells when compared to those of controls. In the spleen, Tp cells were increased, and Ty cells were decreased resulting in an abnormally high Tp/Ty ratio. The abnormally low ratio of Tr/Ty cells in the peripheral blood were associated with poor locomotor responses of T cells toward both chemoattractants. By contrast, abnormally high ratios of TpITy cells in spleens of Hodgkin’s disease were accompanied with increased locomotor responses of T cells toward casein and endotoxin-activated serum when compared with those of peripheral blood from same patients. Immediately following splenectomy, Tp cells in the peripheral blood were increased and locomotor response of T cells increased over presplenectomy levels. These results support the concept of “ecotaxopathy” which might be one of the contributory factors responsible for certain immunodeficiencies observed in patients with Hodgkin’s disease.
INTRODUCTION
Hodgkin’s disease is associated with defective cell-mediated immunity. They include depressed delayed cutaneous hypersensitivity to recall antigens and sensitizing chemical agents such as dinitrochlorobenzene (l-5) and impaired response of peripheral blood lymphocytes to phytohemagglutinin (PHA) and concanavalin A (Con A) (3, 5- 13). The observations regarding proportion of circulating T lymphocytes in Hodgkin’s disease and their relationship with the stage of disease have been rather conflicting (14-20). On the other hand, several studies of the spleen and lymph nodes of Hodgkin’s disease patients demonstrated increased percentages of T cells, especially when they are histologically involved (18, 21-23). These observations along with the finding of increased PHA responsive cells in the spleen from Hodgkin’s disease patients and increased PHA responsive cells in the peripheral blood following splenectomy (23) lead to the hypothesis of maldistribution of lymphoid cells between peripheral blood and spleen in Hodgkin’s disease. Recently, human T lymphocytes have been divided 133 0090- 1229/80/020133- 1 1$01.00/O Copynghl 0 1980 by Academic Press. Inc. All rights of reproduction m any form reserved.
134
CUPI-A AND -r.\h
into at least three subpopulations based on the presence of Fc receptors for IgM(TF), IgG(Ty), or IgA(TLu) (24-27). Functional properties of T/1 and Ty cells have been studied in detail (28, 29). Tp cells populations contain a subpopulation of cells that act as helpers in the differentiation of B cells to plasma cells (not all Tp cells are helpers) and Ty contain cells that act as suppressors in a similar system and mediate antibody-dependent (ADCC) and spontaneous lymphocytemediated (SLMC) cytotoxicity (24, 30). Furthermore. T,u cells respond by proliferation to both Con A and PHA while Ty only respond to Con A (31). Of special interest is our recent demonstration that Tp cells move well toward chemoattractant, casein. and Ty cells either do not move or move poorly toward casein (32). In the present study we have studied lymphocytes from peripheral blood and spleens of untreated children with Hodgkin’s disease with the following aims: (i) To examine the maldistribution of lymphocyte subpopulations particularly of Tp and Ty cells, (ii) to ascertain whether the maldistribution of T-cell subsets is associated with abnormality of locomotion of T cells among two compartments. and (iii) to determined the effect of splenectomy on peripheral blood T-cell subsets and locomotor response of T cells to the chemotactic stimuli. MATERIALS AND METHODS Tissues and blood was obtained from 1I untreated children with Hodgkin’s disease hospitalized at the Memorial Hospital: 16 age- and sex-matched children (diagnosis of gastroenteritis or battered child) served as controls. Spleens were cut in small pieces and passed through a wire mesh to obtain a single-cell suspension. Mononuclear cells were isolated from heparinized peripheral venous blood and splenic cell suspensions on Ficoll-Hypaque gradient. Mononuclear cells were washed 3~ in Hanks’ balanced salt solution (HBSS) and resuspended in medium RPMI-1640 (Grand Island Biological Co.. Grand Island, N.Y.), containing 20% heat-inactivated fetal calf serum (FCS) at a concentration of 4 x 10” cells/ml. A small aliquot was used to assay for conventional surface markers of T. B. and third population cells (33). In this suspension phagocytic cells were labeled by ingestion of 0.X-Frn size polystyrene latex particles (Dow Chemicals. Indianapolis. Ind.). Cells were washed and resuspended in HBSS at 4 x lO”/ml. Tp and Ty cells and T-cell locomotion were assayed on purified T lymphocytes. Purijicutic~tl of’ T L~mplux.ytes Aliquots of mononuclear cells suspended in medium containing FCS were mixed with carbonyl iron (Lymphocyte Separator Reagent Technicon. Tarrytown. N.Y.) at a ratio of 2: I (v/v) and incubated at 37°C for 30 min on a rotator. Phagocytic cells were separated from lymphoid cells on FH density gradient by centrifugation at 400~ for 20 min. Lymphoid cells from the interface were washed 3~ with HBSS and resuspended to a concentration of 4 x lO”/ml. One milliliter of lymphocyte suspension was mixed with 1 ml of neuraminidase-treated I?$ sheep red blood cells (SRBC) and 0.25 ml of heatinactivated FCS (absorbed with SRBC), i.e., 1ymphocyte:SRBC ratio of 1:20. The mixture was incubated at 37°C for 5 min. centrifuged at 200~ for 5 min, followed by incubation on ice for 1hr. The pellets were resuspended and rosetting T cells were separated from non-T cells on FH by centrifugation at 400cqfor 20 min
LOCOMOTION
OF
T CELLS
AND
T-CELL
SUBSETS
IN
HD
135
at 22°C. SRBC attached to T cells were lysed with Tris buffer containing 0.83% ammonium chloride (pH 7.2). Cells were washed 3x with HBSS, resuspended in RPM1 1640 containing 20% heat-inactivated FCS, 100 U/ml penicillin, 100 pg/ml streptomycin, and 2 mM I-glutamine and incubated at 37°C for overnight. T cells were more than 97% purified. Following incubation T cells were washed 3 x with Geys balanced salt solution (GBSS) and resuspended to a concentration of 4 x 10” ml for the analysis of TF and Ty cells and at a concentration 2 x 10Yml for locomotor assay. Viability of the purified T cells was more than 98%. Lymphocyte Subpopulations T lymphocytes were enumerated by spontaneous rosette formation with SRBC (33). Lymphocytes with receptors for IgGFc, Cs, or mouse red blood cells (MRBC) were assayed by rosette techniques using ox RBC-coated IgG (EA), SRBC coated with 19 S IgM antibody and complement (EAC), and fresh nonimmune MRBC (33). The so-called “third population” of lymphoid cells were identified by Ripley rosette test (33). B lymphocytes with surface immunoglobulins were enumerated by the use of fluorescein isothiocyanate-conjugated antiserum [F(ab’),] against all immunoglobulins, polyspecific (PV), and antisera monospecific for p, 6, CY,and y heavy chains of immunoglobulins (33). Subpopulations of .T Lymphocytes Tp and Ty cells were enumerated on purified T-cell population which were incubated overnight at 37°C in medium containing FCS. OxRBC-antibody complexes. IgM and IgG anti-oxRBC antibody were prepared and purified by the technique described (34). OxRBC-antibody complexes were prepared by incubating 2% oxRBC with IgM (1:20 dilution) or IgG (1: 100 dilution) antibody at room temperature for 2 hr. Both oxRBC-IgM (EAm) and oxRBC-IgG (EAg) complexes were washed 3 x in HBSS and resuspended to 1% solution. Complexes were stored at 4°C. T cells with receptors fou ZgM (TpJ OY IgG (T-y). One hundred microliters of T-cell suspension (4 x 106/ml) was mixed with 100 ~1 of 1% EAm (for Tw) or EAg (for Ty), centrifuged at 200g for 5 min and incubated on ice for 1 hr. Pellets were resuspended and 200 T lymphocytes counted for rosette formation. Three or more RBC attached to a T cell was considered a rosette. The results of analysis of Tp and Ty cells are presented as percentage of total T cells. Statistical analysis was done by unpaired Student’s t test. Locomotor Assay. Casein (Mercks, Darmstadt, West Germany) at a concentration of 1 mg/ml and endotoxin-activated serum as 1% solution were used to promote locomotion; Gey’s solution was used as a negative control. In all experiments, filters of 8 pm pore size were used and incubated for 3 hours at 37°C in 5% CO,. The tests were carried out in modified Boyden chambers and cell locomotion was assayed by leading front method (32). RESULTS
The data regarding characteristics of patients are shown in Table 1. Eight of eleven patients had the nodular sclerosis form of H.D., 3 were of mixed cellular type. One patient each were of stages I and II, 6 were of stage III, and 3 of stage IV. Five of eight patients had splenic involvement by H.D. tumor. Absolute
136
GUPTA
AND
T-AN
TABLE CLINICAL
S.N
6 8 9 10 II Patients Controls
AND
HISTOPATHOLOGICAL
Age/Sex
Stage
18/M 16/M 16/F 11/M 14/F 7iM IO/M 3/M 19/F 14/M 17°F
I-A II-B III-A III-A III-A III-B III-B III-B” IV-B IV-B” IV-B”
( 1 I) ( 11)
‘I Refers to clinical staging. (MC) mixed cellularity.
All other
DATA
1 OF CHILDREX
Histopathology MC NS NS NS NS NS MC MC NS NS NS Mean
t SD
patients
were
UITH
Hon
DISEAS~
Absolute lymphocyte (counts/mm,‘)
Spleen involved
1368 4788 I224 3404 3920 6X4 3164 3906 I1 13 627 1740 2449 I 1488 3477 i 1422 P ao.1
pathologically
staged.
(NS)
nodular
sclerosis:
lymphocyte counts in patient groups were comparable to those of controls (P i 0.1). However, 5 of 11 patients had absolute lymphopenia. Lymphocyte
Subpopulations
Results of marker analysis of T, B, and third population lymphoid cells in the peripheral blood are shown in Fig. 1. The proportions of (mean +- SD) T-cell EA rosettes, Ripley rosettes, EAC rosettes, MRFC, and sIgPV-, sIgM-, slgD-, sIgG-, and sIgA-positive lymphocytes in patient group (80.6 + 4.1, 19.7 i 6.8, 9.8 F 2.7. 10.1 L 6.5, 4.7 ? 2.5, 11.2 +- 4.7, 8.3 t 4.3, 5.8 ) 4.6, 2.2 + 2.4, 2.0 ? 2.1. respectively) were comparable to those of the control group (82.0 & 8.0, 23.0 + 7.0, 13.5 t 6.0, 11.0 + 4.0, 6.5 -+ 5.0, 9.8 c 5.0, 7.0 t 3.0, 5.0 z 3.0, 1.2 + 1.2, 1.0 -r- 1.2, respectively). Results of surface markers on mononuclear cells from spleen of five patients are shown in Fig. 2. These results are compared with those of five spleens obtained at other times because of accidental trauma. Properties of T cells in patients were (67.5 L 10.5) significantly higher (P < 0.001) than those of control spleens (35.0 t 8.0). Fc receptor-positive cells (37.0 2 10.9) were significantly decreased (P c: 0.01). when compared with those of controls (59.0 2 6.0). Ripley rosette-forming cells were comparable in two groups. Complement receptor-bearing lymphocytes in patient’s spleens (28.3 & 5.1) were significantly lower (P < 0.01) than those of controls (40.1 t 7.0). MRFC in patient group (13.3 _i 6.6) were lower than those of controls (20.0 +- 4.0). Surface immunoglobulin-bearing B cells (PV, sIgM, sIgD) were (28.8 & 12.9, 22.2 t 12.0, 12.6 & 5.7, respectively) significantly (P < 0.01) lower than those in controls (45.0 t 8.0, 36.0 + 9.0, 28.5 t 7.0, respectively). No significant difference was observed in B cells bearing surface IgG or IgA when compared to those of controls. Data regarding analyses of Tp and T-y cells in peripheral blood are shown in Fig. 3.
LOCOMOTION
OF T CELLS AND T-CELL
q
1
EA
RIPLEY
EAC
MRK
PERIPi4RRAL
FIG. 1. Subpopulations of lymphocytes Hodgkin’s disease (for details see text).
137
SUBSETS IN HD
CONTROL
(16>
PATIENTS
CR)
PV
M
D
G
A
MOOD
in the peripheral
blood of normals and patients with
Proportions of Tp cells (43.0 k 12.0) were comparable to those of controls (48.0 k 11.0). Ty cells were moderately but not significantly increased in patients (20.0 + 10.0) as compared to controls (12.0 ? 5.0). Tp/Ty ratio 2.5 2 1.5 were decreased (P < 0.025) when compared to controls (4.3 r 2.5). Results of TV and Ty cell proportions in the spleen are shown in Fig. 4. Tp cells in the patient group were significantly (P < 0.001) higher (32.0 + 17.0) than those of control group (10.0 t 4.0). Ty cells were significantly (P < 0.001) decreased 19.0 -+ 7.0) when compared to those of controls (45.0 2 10.0). Abnormality of the proportions of TE.Land Ty cells resulted in significantly (P < 0.001) higher ratio of TI.L/T~ cells in the spleen of patients when compared to control spleen.
1
EA
RIPLEY
EAC
MRFC
0
CONTROL (5)
n
PATIENTS (6)
PV
SPLEEN
FIG. 2. Subpopulations of lymphocytes in spleens from normals and patients with Hodgkin’s disease (see text for details).
138
Ty CELLS
b CELLS
PERIPHERAL
FIG. 3. Subpopulations patients with Hodgkin’s
1 I (11 1
BLOOD
of T cells (T/L, Ty. and T/JTy) disease.
in the peripheral
blood
from
normals
and
Data regarding locomotion of T cells are given in Figs. 5 and 6. Peripheral blood T cell migrated toward casein more poorly than control T cells. Response of T cell to chemotactic stimuli of casein was significantly (P < 0.01) poorer than EAS (P c 0.05) when compared to those of control T cells toward these two chemoattractants. Peripheral blood T cells moved more poorly than the T cells from the spleens of same patients (Fig. 6). 0 m
CONTROL PATIENTS
60 z E 50
5
g 2
40
4
I :
30
3f
z 20 Ti
2c
a L
1
0 OL J
10
i
(5) ( ) TM CELLS SPLEEN
FIG. 4. Subpopulations Hodgkin’s disease.
of T cells (Tp.
Ty, and TpITy)
in the spleens
of normals
and patients
with
LOCOMOTION
OF
T CELLS
AND
T-CELL
SUBSETS
El
IN
CONlROl
16
PATIENTS
IO
HD
139
T
GBSS
EAS
CASEIN PERIPHERAL
FIG.
toward (GBSS)
PERIPHERAL
and patients withHodgkin’s disease (EAS). Gey’s balanced salt solution
5
CASEIN HODGKIN’S
6. Locomotion endotoxin-activated
of normals serum
BLO’iXl
GRSS
FIG.
BLOOD
5. Locomotion of T cells from peripheral blood chemoattractants. casein, and endotoxin-activated is a negative control.
EAS
DISEASE
of T cells from peripheral blood and spleens of same patients toward casein serum (EAS) Gey’s balanced salt solution (GBSS) is a negative control.
and
140
GUPTA
EFFECTS OF
FIG. toward
7. Effect of splenectomy casein and endotoxin-activated
Effect
of Splenectom?
AND
I-AN
SCLENKTOMY
on peripheral blood serum (EAS).
T-cell
subsets
and on locomotion
of T cells
T lymphocytes from peripheral blood of three patients were studied within a week following splenectomy for Tp, T-y, and T-cell locomotion. Results are shown in Fig. 7. Proportions of Tp cells increased over presplenectomy levels but had no effect on Ty cells. Significant improvement in the locomotor response of T cells toward casein was observed. However, the effect on locomotion toward EAS was variable. DISCUSSION
Studies of lymphocytes from untreated patients with Hodgkin’s disease have demonstrated both quantitative and qualitative abnormalities. They include lymphopenia, decreased T-cell numbers, depressed it? litro proliferative response to mitogens and antigens, and depressed delayed cutaneous hypersensitivity to recall antigens and sensitizing chemical agents (1 - 131. Increased suppressor activity of adherent cells (35, 36) and certain serum factors (37-40) have been proposed as contributory factors in certain quantitative and functional abnormalities of lymphocytes from patients with Hodgkin’s disease. The concept of “ecotaxopathy” has been proposed based on dichotomy of proliferative responses of lymphocytes to PHA between peripheral blood and spleen (231 and improvement in these responses in the peripheral blood following splenectomy (23) and increased T-cell numbers in the spleen (18, 21-23). In the present study, we have demonstrated absolute lymphopenia in 40% of patients, but with no correlation with histology or stage of disease. A maldistribution of T cells and T-cell subsets between peripheral blood and spleen from patients with untreated Hodgkin’s disease was observed. Furthermore, these changes in T-cell subsets were associated with abnormal locomotor responses of T cell toward chemoattractants. The proportions of peripheral blood T lymphocytes as measured by rosetting with SRBC, in untreated patients with Hodgkin’s disease have been
LOCOMOTION
OF
T CELLS
AND
T-CELL
SUBSETS
IN
HD
141
reported to be decreased (9, 14, 17, 19-38) or normal (15, 16, 18). Bobrove et ul. (9) using anti-T-cell serum found comparable proportion of T cells in patients with Hodgkin’s disease and controls; however, they observed decreased proportions of E-rosette-forming T cells. This discrepancy between these two methods of T-cell enumeration could suggest a selective alteration of binding site on T cells for SRBC. Recently, Fuks et al. (38) have demonstrated the presence of a serum factor in patients with Hodgkin’s disease that interferes with rosetting of T cells with SRBC. Furthermore, incubation of the patients’ mononuclear cells in 20% FCS normalized the proportions of T cells. The exact nature of this serum inhibitor is not known but it has been alleged to be glycoprotein, low-density lipoprotein, or ferritin (39). In our study, we have demonstrated normal proportions of peripheral blood T cells. To identify contaminating phagocytic cells, we use 20% FCS to resuspend mononuclear cells and incubated them with latex particles at 37°C for 30 min. It is likely that any serum inhibitory factor is released during this short period of incubation. It, therefore, appears that the relative number of peripheral blood T lymphocytes in Hodgkin’s disease are normal or close to normal while SRBC binding T lymphocytes may be depressed due to interaction of lymphocyte surface receptors with serum factors. Proportion or number of T cells in our study did not correlate with the histopathology or clinical stage of disease. A number of investigators have reported a high proportion of T lymphocytes in spleens from Hodgkin’s disease (21-23). The observation along with normal PHA response in the spleens and depressed PHA-responsiveness cells in the peripheral of Hodgkin’s disease favor the concept of sequestration of at least one subpopulation of T cells in the spleen of Hodgkin’s disease. Our present study also demonstrated increased proportions of T cells in the spleens from Hodgkin’s disease. Furthermore, in this study we have demonstrated increased proportions of Ty cells in the peripheral blood and increased proportions of Tp cells in the spleens of Hodgkin’s disease. Tp cells have shown to respond to proliferation to PHA while Ty cells do not respond (31). Our observations of increased Ty cells in peripheral blood and increased Tp cells in the spleens could explain the decreased peripheral blood lymphocyte response to PHA and normal PHA response in spleens of Hodgkin’s disease reported by de Sousa et al. (23). B lymphocytes, as defined by the presence of surface immunoglobulin and receptor for complement, are normal or slightly decreased in untreated patients with Hodgkin’s disease (5, 16, 15, 18). In the present study we have also demonstrated normal proportions of B cells as defined by the presence of surface immunoglobulin using F(ab)‘, antisera specific against all classes of Ig and monospecific against II, 6, y, and CKheavy chains, receptors for MRBC, and Cs receptors. The numbers of high-affinity, Fc-receptor-bearing third population cells were also comparable to a control group, a finding that is further supported by normal natural killing and antibody-dependent cytotoxic activity in adult patients with Hodgkin’s disease (41). In this study B lymphocytes (MRFC, SIg(PV), SIgM, SIgD, and EAC) were decreased in the spleens of patients with Hodgkin’s disease. Hunter ef al. (42) reported increased proportions of mononuclear cells with IgMFc receptors in the spleens of Hodgkin’s disease. However, these investigators did not define the cell population having IgMFc receptors, furthermore, they did not incubate the cells in FCS at 37”C, a procedure which is required for
142
CUP-IA
AND
L.4N
the full expression of IgM receptors on Tp cells (34). Romagnani rf rrl. (43) observed increased proportions of Ty cells and decreased proportions of Tp cells in the peripheral blood from patients with Hodgkin’s disease. These investigators. however, did not examine T-cell subsets simultaneously in the peripheral blood and spleens. Our study demonstrated increased Ty cells in the peripheral blood and increased Tp cells and decreased Ty-cell proportions in the spleens resulting in an abnormal ratio of Tp/Ty cells both in the peripheral blood and spleens. Recently, analyzing locomotor activity of lymphocytes in Boyden chambers we have demonstrated that Tp cells migrate well and Ty cells migrate poorly toward chemoattractants. Therefore, we examined the locomotion of T cells in the peripheral blood and spleens from these patients with childhood Hodgkin’s disease to determine whether abnormal distribution of T-cell subsets between spleen and peripheral blood is associated with abnormality of locomotion. Peripheral blood T cells of patients with Hodgkin’s disease moved much more poorly than those of control peripheral blood T cells. Splenic T cells from patients moved better than their peripheral blood T cells. Following splenectomy. proportions of Tp cells increased in the peripheral blood and locomotion of T cells particularly toward casein improved over presplenectomy levels. DeSousa ct trl. (23) have reported improvement in blood lymphocyte PHA response following splenectomy. In summary. our study of untreated children with Hodgkin‘s disease demonstrates an abnormal distribution of T cells and T-cell subsets between peripheral blood and spleens accompanied with abnormal locomotion of T cells. Furthermore, these abnormalities return toward normal immediately following splenectomy. These observations further support the concept of “ecotaxopathy” which could be one of the determining factors in the pathogenesis of certain immunodeficiencies observed in Hodgkin’s disease. AKNOWLEDGMENTS We wish to thank Dr. Robert Good for his critical and valuable comments. The expert skillful technical assistance of Ms. S. Khanna, Ms. E. Frederick, Ms. S. Garber. and Mr. A. Prajapati is gratefully acknowledged. This work is supported by National Institutes of Health Grants CA-17404. CA-19267. CA-23766. CA-08748, AI-11843, NS-I 1457. and AG-00541, Funds for the Advanced Study of Cancer. and Judith Harris Selig Memorial Fund.
REFERENCES I. Eltringham, J. R.. and Kaplan. H. S., C‘crnc (‘Y /tr\r. Mcur~~y. 36, 107. 1973. 2. Young. R. C.. Corder, M. P., Berad, C. W.. and DeVita. V. ‘I’.. .40rrr. ./. nfc(I. 52. 63. 1972. 3. Helm. G.. Mellstedt. H.. Bjorkholm. M., Johansson, B.. Killander. D.. Sundblad, R., and Soderberg, G., Ctrncer 37. 751. 1976. 4. Aisenberg. A. C.. J. C/i/i. f~i,c~sr. 41, 1964. 1962. 5. Chase. D. C. Jr.. Hansen, J. A.. Corrales. E.. Young. C. W.. DuPont, B., Pinsky. C. M.. and Good. R. A., C‘rrn<,c~r 38. 1807, 1976. 6. Ziegler, J. B.. Hansen. P.. and Penny. R., C/i/i. Inrnrrl,irj/. /nr,,?~,,,c~ptrth~,i. 3, 451. 1975. 7. Twomey, J. J.. Laughter, A. H.. Lazar. S., and Donglass, C. C.. C~r!c,rr 38, 740. 1976. 8. Han. T., and Sokal. J. R.. Amer. J. Med. 48. 728. 1970. 9. Bobrove. A. M., Fuks. Z.. Strober. S.. and Kaplan. H. S.. C‘
LOCOMOTION
OF T CELLS AND T-CELL
SUBSETS IN HD
143
13. Romagnani, S., Amadori, A., Biti, G., Bellesi, G., and Ricci, M., Int. Arch. Allergy Appl. Irnmunol. 51, 378, 1976. 14. Bukowski, R. M., Noguchi, S., Hewlett, .I. S.. and Deodhar, S., Amer. J. Clin. Path/. 65, 31, 1976. IS. Heier, H. E., and Normann, T., Stand. J. Hrmarol. 13, 199, 1974. 16. Gajl-Peczalska, K. J., Bloomfield, C. D., Sosin, H., and Kersey, J. H., C&I. Exp. /mmund. 23, 47, 1976. 17. Andersen, E.. Stand. .I. Hemarol. 12, 263. 1974. 18. de Sousa, M., Tan, C. T. C., Siegal, F. D., Filippa, D. A., Tan, R., and Good, R. A., Ped. Res. 12, 143, 1978. 19. Bensa, J. C., Micouin, C., Schaerer, R., Sotto, J. J., and Holland, D., Biomedicinp 26, 137. 1977. 20. Aisenberg, A. C., Wetzman, S., and Wilkes, B.. Blood 54, 439, 1978. 21. Payne, S. V.. Jones, D. B.. Galgart. D. G., Smith, J. C., and Wright, D. H., Clirr. Exp. Immunol. 24, 280, 1976. 22. Kaur, J.. Catovsky, D., Spiers, A. S. D., and Galton, D. A. G., Lancer 1, 834, 1974. 23. de Sousa, M., Yang, M., Lopez-Corrales, E., Tan. C., Hausen, J. A., DuPont, B.. and Good, R. A., C/in. E.YP. Immunol. 27, 143, 1977. 24. Moretta L., Webb, S. R.. Grossi, C. E., Lydyard, P. M., and Cooper, M. D., J. hp. Med. 146, 184, 1977. 25. Gupta, S., Platsoucas, C. D., Schulof, R., and Good, R., Ceil fmmunol., 45, 469, 1979. 26. Gupta, S., Platsoucas, C. D., and Good, R. A., Proc. Nar. Arad. SC;. USA, 76, 4025, 1979. 27. Lum. L. G., Muchmore, A. V., Keren, D., Dicker, J., Koski. Stober, W., and Blaese, R. M.,J. Immrtnol. 120, 1278, 1978. 28. Gupta, S.. C/in. Bull. 8, 100, 1978. 29. Gupta. A., and Good, R. A., In “Human Lymphocyte Differentiation. Its Application to Cancer.” (B. Serrou and C. Rosenfeld, Eds.), p. 367, North-Holland, Amsterdam, 1978. 30. Gupta. S., Fernandes, G., Nair, M., and Good, R. A.. Acad. Sci. USA 75, 5137, 1978. 31. Moretta, L., Ferrarini, M., Migari, M. C., Moretta, A., and Webb, S. R.,J. Immune/. 17, 2171, 1976. 32. Parrott. D. M. V., Good, R. A., O’Neill, G. J., and Gupta. S., Proc. Nat. Acad, Sci. USA 75,2392, 1978. 33. Gupta, S., Good, R. A., and Siegal, F. P., C/in. Exp. Immunol. 25, 319, 1976. 34. Gupta. S., and Good, R. A., Cell. Immunol. 34, 10, 1977. 35. Goodwin, J. S., Messner, R. P., Bankhurst, A. D.. Peake, G. T., Saiki, J. H., and Williams, R. C.. N. Engl. J. Med. 297, 963, 1977. 36. Sibbitt, W. L., Bankhurst, A. D., and Williams, R. C., J. C/in. Inrfesr. 61, 55, 1978. 37. Moroz, C., Lahat, N., Biniaminov, M.. and Ramot, B., C’lin. Exp. Immunol. 29, 30, 1977. 38. Fuks, Z., Strober, S., and Kaplan, H. S., N. Engl. .I. Med. 295, 1273, 1976. 39. Siegal. F. P.. Neti, Engl. J. Med. 295, 1313, 1976. 40. Ahmol, P. L., and Unger. A., C/in. Exp. Immunol. 26, 520, 1976. 41. Fernandes, G., Schulof, R., Nair, M., Straus, D., Lee, B., Clarkson, B., Good, R. A., and Gupta, S., “13th Intl. Leukocyte Culture Conf., Ottawa,” p. 121, May 22-25, 1979. 42. Hunter. C. P.. Pincus. G.. Woodward, L., Moloney, W. C., and Churchill, W. M.. Cc//. Immunol. 31, 193. 1977. 43. Romagnani. S., Maggi, E., and Biagiotti, R., Giudizi, Amadori, A., and Ricci, M., J. Immunol. 7, 511. 1978.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
Subpopulations
15,
133-143 (1980)
of Human
T Lymphocytes
XIV. Abnormality of T-Cell Locomotion and of Distribution of Subpopulations of T and 6 Lymphocytes in Peripheral Blood and Spleen from Children with Untreated Hodgkin’s Disease SUDHIR Memoriul
Sloan-Kettering
GUPTA Cancer
AND CHARLOTTE Center.
New
York.
TAN New,
York
10021
Received June 13. 1979 Peripheral blood and splenic mononuclear cells from untreated children with Hodgkin’s disease were examined for the proportions of T, B, and third population cells, T cells with IgM(Tp) or IgG(Ty1 receptors, locomotion of T cells toward chemoattractants, casein- and endotoxin-activated serum, and for the effect of splenectomy on T-cell subsets and on locomotion of T cells. In the peripheral blood, proportions of T, B, and third population cells were comparable to those of controls, however, spleen contained increased proportions of T cells and decreased proportion of B lymphocytes. TF cells were comparable and Ty cells were moderately increased in the peripheral blood resulting in an abnormally low ratio of T&/Ty cells when compared to those of controls. In the spleen, Tp cells were increased, and Ty cells were decreased resulting in an abnormally high Tp/Ty ratio. The abnormally low ratio of Tr/Ty cells in the peripheral blood were associated with poor locomotor responses of T cells toward both chemoattractants. By contrast, abnormally high ratios of TpITy cells in spleens of Hodgkin’s disease were accompanied with increased locomotor responses of T cells toward casein and endotoxin-activated serum when compared with those of peripheral blood from same patients. Immediately following splenectomy, Tp cells in the peripheral blood were increased and locomotor response of T cells increased over presplenectomy levels. These results support the concept of “ecotaxopathy” which might be one of the contributory factors responsible for certain immunodeficiencies observed in patients with Hodgkin’s disease.
INTRODUCTION
Hodgkin’s disease is associated with defective cell-mediated immunity. They include depressed delayed cutaneous hypersensitivity to recall antigens and sensitizing chemical agents such as dinitrochlorobenzene (l-5) and impaired response of peripheral blood lymphocytes to phytohemagglutinin (PHA) and concanavalin A (Con A) (3, 5- 13). The observations regarding proportion of circulating T lymphocytes in Hodgkin’s disease and their relationship with the stage of disease have been rather conflicting (14-20). On the other hand, several studies of the spleen and lymph nodes of Hodgkin’s disease patients demonstrated increased percentages of T cells, especially when they are histologically involved (18, 21-23). These observations along with the finding of increased PHA responsive cells in the spleen from Hodgkin’s disease patients and increased PHA responsive cells in the peripheral blood following splenectomy (23) lead to the hypothesis of maldistribution of lymphoid cells between peripheral blood and spleen in Hodgkin’s disease. Recently, human T lymphocytes have been divided 133 0090- 1229/80/020133- 1 1$01.00/O Copynghl 0 1980 by Academic Press. Inc. All rights of reproduction m any form reserved.
134
CUPI-A AND -r.\h
into at least three subpopulations based on the presence of Fc receptors for IgM(TF), IgG(Ty), or IgA(TLu) (24-27). Functional properties of T/1 and Ty cells have been studied in detail (28, 29). Tp cells populations contain a subpopulation of cells that act as helpers in the differentiation of B cells to plasma cells (not all Tp cells are helpers) and Ty contain cells that act as suppressors in a similar system and mediate antibody-dependent (ADCC) and spontaneous lymphocytemediated (SLMC) cytotoxicity (24, 30). Furthermore. T,u cells respond by proliferation to both Con A and PHA while Ty only respond to Con A (31). Of special interest is our recent demonstration that Tp cells move well toward chemoattractant, casein. and Ty cells either do not move or move poorly toward casein (32). In the present study we have studied lymphocytes from peripheral blood and spleens of untreated children with Hodgkin’s disease with the following aims: (i) To examine the maldistribution of lymphocyte subpopulations particularly of Tp and Ty cells, (ii) to ascertain whether the maldistribution of T-cell subsets is associated with abnormality of locomotion of T cells among two compartments. and (iii) to determined the effect of splenectomy on peripheral blood T-cell subsets and locomotor response of T cells to the chemotactic stimuli. MATERIALS AND METHODS Tissues and blood was obtained from 1I untreated children with Hodgkin’s disease hospitalized at the Memorial Hospital: 16 age- and sex-matched children (diagnosis of gastroenteritis or battered child) served as controls. Spleens were cut in small pieces and passed through a wire mesh to obtain a single-cell suspension. Mononuclear cells were isolated from heparinized peripheral venous blood and splenic cell suspensions on Ficoll-Hypaque gradient. Mononuclear cells were washed 3~ in Hanks’ balanced salt solution (HBSS) and resuspended in medium RPMI-1640 (Grand Island Biological Co.. Grand Island, N.Y.), containing 20% heat-inactivated fetal calf serum (FCS) at a concentration of 4 x 10” cells/ml. A small aliquot was used to assay for conventional surface markers of T. B. and third population cells (33). In this suspension phagocytic cells were labeled by ingestion of 0.X-Frn size polystyrene latex particles (Dow Chemicals. Indianapolis. Ind.). Cells were washed and resuspended in HBSS at 4 x lO”/ml. Tp and Ty cells and T-cell locomotion were assayed on purified T lymphocytes. Purijicutic~tl of’ T L~mplux.ytes Aliquots of mononuclear cells suspended in medium containing FCS were mixed with carbonyl iron (Lymphocyte Separator Reagent Technicon. Tarrytown. N.Y.) at a ratio of 2: I (v/v) and incubated at 37°C for 30 min on a rotator. Phagocytic cells were separated from lymphoid cells on FH density gradient by centrifugation at 400~ for 20 min. Lymphoid cells from the interface were washed 3~ with HBSS and resuspended to a concentration of 4 x lO”/ml. One milliliter of lymphocyte suspension was mixed with 1 ml of neuraminidase-treated I?$ sheep red blood cells (SRBC) and 0.25 ml of heatinactivated FCS (absorbed with SRBC), i.e., 1ymphocyte:SRBC ratio of 1:20. The mixture was incubated at 37°C for 5 min. centrifuged at 200~ for 5 min, followed by incubation on ice for 1hr. The pellets were resuspended and rosetting T cells were separated from non-T cells on FH by centrifugation at 400cqfor 20 min
LOCOMOTION
OF
T CELLS
AND
T-CELL
SUBSETS
IN
HD
135
at 22°C. SRBC attached to T cells were lysed with Tris buffer containing 0.83% ammonium chloride (pH 7.2). Cells were washed 3x with HBSS, resuspended in RPM1 1640 containing 20% heat-inactivated FCS, 100 U/ml penicillin, 100 pg/ml streptomycin, and 2 mM I-glutamine and incubated at 37°C for overnight. T cells were more than 97% purified. Following incubation T cells were washed 3 x with Geys balanced salt solution (GBSS) and resuspended to a concentration of 4 x 10” ml for the analysis of TF and Ty cells and at a concentration 2 x 10Yml for locomotor assay. Viability of the purified T cells was more than 98%. Lymphocyte Subpopulations T lymphocytes were enumerated by spontaneous rosette formation with SRBC (33). Lymphocytes with receptors for IgGFc, Cs, or mouse red blood cells (MRBC) were assayed by rosette techniques using ox RBC-coated IgG (EA), SRBC coated with 19 S IgM antibody and complement (EAC), and fresh nonimmune MRBC (33). The so-called “third population” of lymphoid cells were identified by Ripley rosette test (33). B lymphocytes with surface immunoglobulins were enumerated by the use of fluorescein isothiocyanate-conjugated antiserum [F(ab’),] against all immunoglobulins, polyspecific (PV), and antisera monospecific for p, 6, CY,and y heavy chains of immunoglobulins (33). Subpopulations of .T Lymphocytes Tp and Ty cells were enumerated on purified T-cell population which were incubated overnight at 37°C in medium containing FCS. OxRBC-antibody complexes. IgM and IgG anti-oxRBC antibody were prepared and purified by the technique described (34). OxRBC-antibody complexes were prepared by incubating 2% oxRBC with IgM (1:20 dilution) or IgG (1: 100 dilution) antibody at room temperature for 2 hr. Both oxRBC-IgM (EAm) and oxRBC-IgG (EAg) complexes were washed 3 x in HBSS and resuspended to 1% solution. Complexes were stored at 4°C. T cells with receptors fou ZgM (TpJ OY IgG (T-y). One hundred microliters of T-cell suspension (4 x 106/ml) was mixed with 100 ~1 of 1% EAm (for Tw) or EAg (for Ty), centrifuged at 200g for 5 min and incubated on ice for 1 hr. Pellets were resuspended and 200 T lymphocytes counted for rosette formation. Three or more RBC attached to a T cell was considered a rosette. The results of analysis of Tp and Ty cells are presented as percentage of total T cells. Statistical analysis was done by unpaired Student’s t test. Locomotor Assay. Casein (Mercks, Darmstadt, West Germany) at a concentration of 1 mg/ml and endotoxin-activated serum as 1% solution were used to promote locomotion; Gey’s solution was used as a negative control. In all experiments, filters of 8 pm pore size were used and incubated for 3 hours at 37°C in 5% CO,. The tests were carried out in modified Boyden chambers and cell locomotion was assayed by leading front method (32). RESULTS
The data regarding characteristics of patients are shown in Table 1. Eight of eleven patients had the nodular sclerosis form of H.D., 3 were of mixed cellular type. One patient each were of stages I and II, 6 were of stage III, and 3 of stage IV. Five of eight patients had splenic involvement by H.D. tumor. Absolute
136
GUPTA
AND
T-AN
TABLE CLINICAL
S.N
6 8 9 10 II Patients Controls
AND
HISTOPATHOLOGICAL
Age/Sex
Stage
18/M 16/M 16/F 11/M 14/F 7iM IO/M 3/M 19/F 14/M 17°F
I-A II-B III-A III-A III-A III-B III-B III-B” IV-B IV-B” IV-B”
( 1 I) ( 11)
‘I Refers to clinical staging. (MC) mixed cellularity.
All other
DATA
1 OF CHILDREX
Histopathology MC NS NS NS NS NS MC MC NS NS NS Mean
t SD
patients
were
UITH
Hon
DISEAS~
Absolute lymphocyte (counts/mm,‘)
Spleen involved
1368 4788 I224 3404 3920 6X4 3164 3906 I1 13 627 1740 2449 I 1488 3477 i 1422 P ao.1
pathologically
staged.
(NS)
nodular
sclerosis:
lymphocyte counts in patient groups were comparable to those of controls (P i 0.1). However, 5 of 11 patients had absolute lymphopenia. Lymphocyte
Subpopulations
Results of marker analysis of T, B, and third population lymphoid cells in the peripheral blood are shown in Fig. 1. The proportions of (mean +- SD) T-cell EA rosettes, Ripley rosettes, EAC rosettes, MRFC, and sIgPV-, sIgM-, slgD-, sIgG-, and sIgA-positive lymphocytes in patient group (80.6 + 4.1, 19.7 i 6.8, 9.8 F 2.7. 10.1 L 6.5, 4.7 ? 2.5, 11.2 +- 4.7, 8.3 t 4.3, 5.8 ) 4.6, 2.2 + 2.4, 2.0 ? 2.1. respectively) were comparable to those of the control group (82.0 & 8.0, 23.0 + 7.0, 13.5 t 6.0, 11.0 + 4.0, 6.5 -+ 5.0, 9.8 c 5.0, 7.0 t 3.0, 5.0 z 3.0, 1.2 + 1.2, 1.0 -r- 1.2, respectively). Results of surface markers on mononuclear cells from spleen of five patients are shown in Fig. 2. These results are compared with those of five spleens obtained at other times because of accidental trauma. Properties of T cells in patients were (67.5 L 10.5) significantly higher (P < 0.001) than those of control spleens (35.0 t 8.0). Fc receptor-positive cells (37.0 2 10.9) were significantly decreased (P c: 0.01). when compared with those of controls (59.0 2 6.0). Ripley rosette-forming cells were comparable in two groups. Complement receptor-bearing lymphocytes in patient’s spleens (28.3 & 5.1) were significantly lower (P < 0.01) than those of controls (40.1 t 7.0). MRFC in patient group (13.3 _i 6.6) were lower than those of controls (20.0 +- 4.0). Surface immunoglobulin-bearing B cells (PV, sIgM, sIgD) were (28.8 & 12.9, 22.2 t 12.0, 12.6 & 5.7, respectively) significantly (P < 0.01) lower than those in controls (45.0 t 8.0, 36.0 + 9.0, 28.5 t 7.0, respectively). No significant difference was observed in B cells bearing surface IgG or IgA when compared to those of controls. Data regarding analyses of Tp and T-y cells in peripheral blood are shown in Fig. 3.
LOCOMOTION
OF T CELLS AND T-CELL
q
1
EA
RIPLEY
EAC
MRK
PERIPi4RRAL
FIG. 1. Subpopulations of lymphocytes Hodgkin’s disease (for details see text).
137
SUBSETS IN HD
CONTROL
(16>
PATIENTS
CR)
PV
M
D
G
A
MOOD
in the peripheral
blood of normals and patients with
Proportions of Tp cells (43.0 k 12.0) were comparable to those of controls (48.0 k 11.0). Ty cells were moderately but not significantly increased in patients (20.0 + 10.0) as compared to controls (12.0 ? 5.0). Tp/Ty ratio 2.5 2 1.5 were decreased (P < 0.025) when compared to controls (4.3 r 2.5). Results of TV and Ty cell proportions in the spleen are shown in Fig. 4. Tp cells in the patient group were significantly (P < 0.001) higher (32.0 + 17.0) than those of control group (10.0 t 4.0). Ty cells were significantly (P < 0.001) decreased 19.0 -+ 7.0) when compared to those of controls (45.0 2 10.0). Abnormality of the proportions of TE.Land Ty cells resulted in significantly (P < 0.001) higher ratio of TI.L/T~ cells in the spleen of patients when compared to control spleen.
1
EA
RIPLEY
EAC
MRFC
0
CONTROL (5)
n
PATIENTS (6)
PV
SPLEEN
FIG. 2. Subpopulations of lymphocytes in spleens from normals and patients with Hodgkin’s disease (see text for details).
138
Ty CELLS
b CELLS
PERIPHERAL
FIG. 3. Subpopulations patients with Hodgkin’s
1 I (11 1
BLOOD
of T cells (T/L, Ty. and T/JTy) disease.
in the peripheral
blood
from
normals
and
Data regarding locomotion of T cells are given in Figs. 5 and 6. Peripheral blood T cell migrated toward casein more poorly than control T cells. Response of T cell to chemotactic stimuli of casein was significantly (P < 0.01) poorer than EAS (P c 0.05) when compared to those of control T cells toward these two chemoattractants. Peripheral blood T cells moved more poorly than the T cells from the spleens of same patients (Fig. 6). 0 m
CONTROL PATIENTS
60 z E 50
5
g 2
40
4
I :
30
3f
z 20 Ti
2c
a L
1
0 OL J
10
i
(5) ( ) TM CELLS SPLEEN
FIG. 4. Subpopulations Hodgkin’s disease.
of T cells (Tp.
Ty, and TpITy)
in the spleens
of normals
and patients
with
LOCOMOTION
OF
T CELLS
AND
T-CELL
SUBSETS
El
IN
CONlROl
16
PATIENTS
IO
HD
139
T
GBSS
EAS
CASEIN PERIPHERAL
FIG.
toward (GBSS)
PERIPHERAL
and patients withHodgkin’s disease (EAS). Gey’s balanced salt solution
5
CASEIN HODGKIN’S
6. Locomotion endotoxin-activated
of normals serum
BLO’iXl
GRSS
FIG.
BLOOD
5. Locomotion of T cells from peripheral blood chemoattractants. casein, and endotoxin-activated is a negative control.
EAS
DISEASE
of T cells from peripheral blood and spleens of same patients toward casein serum (EAS) Gey’s balanced salt solution (GBSS) is a negative control.
and
140
GUPTA
EFFECTS OF
FIG. toward
7. Effect of splenectomy casein and endotoxin-activated
Effect
of Splenectom?
AND
I-AN
SCLENKTOMY
on peripheral blood serum (EAS).
T-cell
subsets
and on locomotion
of T cells
T lymphocytes from peripheral blood of three patients were studied within a week following splenectomy for Tp, T-y, and T-cell locomotion. Results are shown in Fig. 7. Proportions of Tp cells increased over presplenectomy levels but had no effect on Ty cells. Significant improvement in the locomotor response of T cells toward casein was observed. However, the effect on locomotion toward EAS was variable. DISCUSSION
Studies of lymphocytes from untreated patients with Hodgkin’s disease have demonstrated both quantitative and qualitative abnormalities. They include lymphopenia, decreased T-cell numbers, depressed it? litro proliferative response to mitogens and antigens, and depressed delayed cutaneous hypersensitivity to recall antigens and sensitizing chemical agents (1 - 131. Increased suppressor activity of adherent cells (35, 36) and certain serum factors (37-40) have been proposed as contributory factors in certain quantitative and functional abnormalities of lymphocytes from patients with Hodgkin’s disease. The concept of “ecotaxopathy” has been proposed based on dichotomy of proliferative responses of lymphocytes to PHA between peripheral blood and spleen (231 and improvement in these responses in the peripheral blood following splenectomy (23) and increased T-cell numbers in the spleen (18, 21-23). In the present study, we have demonstrated absolute lymphopenia in 40% of patients, but with no correlation with histology or stage of disease. A maldistribution of T cells and T-cell subsets between peripheral blood and spleen from patients with untreated Hodgkin’s disease was observed. Furthermore, these changes in T-cell subsets were associated with abnormal locomotor responses of T cell toward chemoattractants. The proportions of peripheral blood T lymphocytes as measured by rosetting with SRBC, in untreated patients with Hodgkin’s disease have been
LOCOMOTION
OF
T CELLS
AND
T-CELL
SUBSETS
IN
HD
141
reported to be decreased (9, 14, 17, 19-38) or normal (15, 16, 18). Bobrove et ul. (9) using anti-T-cell serum found comparable proportion of T cells in patients with Hodgkin’s disease and controls; however, they observed decreased proportions of E-rosette-forming T cells. This discrepancy between these two methods of T-cell enumeration could suggest a selective alteration of binding site on T cells for SRBC. Recently, Fuks et al. (38) have demonstrated the presence of a serum factor in patients with Hodgkin’s disease that interferes with rosetting of T cells with SRBC. Furthermore, incubation of the patients’ mononuclear cells in 20% FCS normalized the proportions of T cells. The exact nature of this serum inhibitor is not known but it has been alleged to be glycoprotein, low-density lipoprotein, or ferritin (39). In our study, we have demonstrated normal proportions of peripheral blood T cells. To identify contaminating phagocytic cells, we use 20% FCS to resuspend mononuclear cells and incubated them with latex particles at 37°C for 30 min. It is likely that any serum inhibitory factor is released during this short period of incubation. It, therefore, appears that the relative number of peripheral blood T lymphocytes in Hodgkin’s disease are normal or close to normal while SRBC binding T lymphocytes may be depressed due to interaction of lymphocyte surface receptors with serum factors. Proportion or number of T cells in our study did not correlate with the histopathology or clinical stage of disease. A number of investigators have reported a high proportion of T lymphocytes in spleens from Hodgkin’s disease (21-23). The observation along with normal PHA response in the spleens and depressed PHA-responsiveness cells in the peripheral of Hodgkin’s disease favor the concept of sequestration of at least one subpopulation of T cells in the spleen of Hodgkin’s disease. Our present study also demonstrated increased proportions of T cells in the spleens from Hodgkin’s disease. Furthermore, in this study we have demonstrated increased proportions of Ty cells in the peripheral blood and increased proportions of Tp cells in the spleens of Hodgkin’s disease. Tp cells have shown to respond to proliferation to PHA while Ty cells do not respond (31). Our observations of increased Ty cells in peripheral blood and increased Tp cells in the spleens could explain the decreased peripheral blood lymphocyte response to PHA and normal PHA response in spleens of Hodgkin’s disease reported by de Sousa et al. (23). B lymphocytes, as defined by the presence of surface immunoglobulin and receptor for complement, are normal or slightly decreased in untreated patients with Hodgkin’s disease (5, 16, 15, 18). In the present study we have also demonstrated normal proportions of B cells as defined by the presence of surface immunoglobulin using F(ab)‘, antisera specific against all classes of Ig and monospecific against II, 6, y, and CKheavy chains, receptors for MRBC, and Cs receptors. The numbers of high-affinity, Fc-receptor-bearing third population cells were also comparable to a control group, a finding that is further supported by normal natural killing and antibody-dependent cytotoxic activity in adult patients with Hodgkin’s disease (41). In this study B lymphocytes (MRFC, SIg(PV), SIgM, SIgD, and EAC) were decreased in the spleens of patients with Hodgkin’s disease. Hunter ef al. (42) reported increased proportions of mononuclear cells with IgMFc receptors in the spleens of Hodgkin’s disease. However, these investigators did not define the cell population having IgMFc receptors, furthermore, they did not incubate the cells in FCS at 37”C, a procedure which is required for
142
CUP-IA
AND
L.4N
the full expression of IgM receptors on Tp cells (34). Romagnani rf rrl. (43) observed increased proportions of Ty cells and decreased proportions of Tp cells in the peripheral blood from patients with Hodgkin’s disease. These investigators. however, did not examine T-cell subsets simultaneously in the peripheral blood and spleens. Our study demonstrated increased Ty cells in the peripheral blood and increased Tp cells and decreased Ty-cell proportions in the spleens resulting in an abnormal ratio of Tp/Ty cells both in the peripheral blood and spleens. Recently, analyzing locomotor activity of lymphocytes in Boyden chambers we have demonstrated that Tp cells migrate well and Ty cells migrate poorly toward chemoattractants. Therefore, we examined the locomotion of T cells in the peripheral blood and spleens from these patients with childhood Hodgkin’s disease to determine whether abnormal distribution of T-cell subsets between spleen and peripheral blood is associated with abnormality of locomotion. Peripheral blood T cells of patients with Hodgkin’s disease moved much more poorly than those of control peripheral blood T cells. Splenic T cells from patients moved better than their peripheral blood T cells. Following splenectomy. proportions of Tp cells increased in the peripheral blood and locomotion of T cells particularly toward casein improved over presplenectomy levels. DeSousa ct trl. (23) have reported improvement in blood lymphocyte PHA response following splenectomy. In summary. our study of untreated children with Hodgkin‘s disease demonstrates an abnormal distribution of T cells and T-cell subsets between peripheral blood and spleens accompanied with abnormal locomotion of T cells. Furthermore, these abnormalities return toward normal immediately following splenectomy. These observations further support the concept of “ecotaxopathy” which could be one of the determining factors in the pathogenesis of certain immunodeficiencies observed in Hodgkin’s disease. AKNOWLEDGMENTS We wish to thank Dr. Robert Good for his critical and valuable comments. The expert skillful technical assistance of Ms. S. Khanna, Ms. E. Frederick, Ms. S. Garber. and Mr. A. Prajapati is gratefully acknowledged. This work is supported by National Institutes of Health Grants CA-17404. CA-19267. CA-23766. CA-08748, AI-11843, NS-I 1457. and AG-00541, Funds for the Advanced Study of Cancer. and Judith Harris Selig Memorial Fund.
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