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Contrasting effects of cyclophosphamide and prednisolone on the phenotype of human peripheral blood leukocytes
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
28, 101-114 (1983)
Contrasting Effects of Cyclophosphamide on the Phenotype of Human Peripheral ROBERT Sidney
C. BAST,
Farber
Cancer
and Prednisolone Blood Leukocytes
JR., ELLIS L. REINHERZ, CAROLE AND STUART F. SCHLOSSMAN Institute,
Brigham Boston,
and Women’s Massachusetts
Hospital, 02115
MAVER, and Harvard
PHILIP Medical
LAVIN, School,
The cell surface phenotype of human peripheral blood mononuclear cells has been characterized before and after intravenous injection of cyclophosphamide or prednisolone. Low doses of cyclophosphamide (100-600 mg/m*) temporarily decrease levels of circulating B lymphocytes. Slightly higher doses of cyclophosphamide (200-600 mg/m”) produce transient depression of TS-, Ml-, and Ia-positive cells. After doses of 200400 mg cyclophosphamide/mz, TCpositive cells are spared, resulting in a transient elevation of the T4fT8 ratio. With higher doses of cyclophosphamide (2 600 mg/m”), all T cells are affected and the T4/T8 ratio declines to pretreatment levels. By contrast, intravenous injection of prednisolone at 40 mg/m* reduces the T4/T8 ratio. Levels of both T4 and TS cells decline, but T4 cells are affected more markedly than TS cells.
INTRODUCTION
The immunosuppressive effects of cyclophosphamide have been documented in animal systems and in clinical studies (1, 2). Inhibition of antibody synthesis has been achieved more readily than inhibition of delayed hypersensitivity after administration of the alkylator. In earlier reports both B cells and T cells decrease in human peripheral blood after cyclophosphamide injection. Selective depletion of B cells has, however, been observed in animal systems (3,4) where low doses of cyclophosphamide have potentiated delayed cutaneous reactivity to protein antigens (5) and contact allergens (6). Pretreatment with cyclophosphamide has eliminated tumor-specific suppressors in murine systems (7) and potentiated the therapeutic effect of immune T lymphocytes (8). At higher dosages, however, both the primary and the anamnestic response to tumor growth can be inhibited (9). Administration of glucocorticoids affects both the distribution and function of human lymphocytes (10). Maximal lymphopenia is observed 4-6 hr after steroid injections and lymphocyte levels return to the normal range in 24-48 hr. T cells are said to be affected more than B cells. Most studies have, however, measured E-rosette-forming cells and it is possible that glucocorticoids have affected expression of erythrocyte receptors rather than the actual number of circulating T cells. When T-cell subsets have been measured after injection of prednisolone, TM cells have been affected more markedly than TG cells in vivo (11). When studied in vitro, only the suppressor function attributed to TG cells has been inhibited (12).
The availability of monoclonal reagents which recognize subsets of T cells has permitted more precise analysis of the impact of cyclophosphamide and predniso101 0090-1229/83 $1.50 Copyrkht 0 1983 by Academic Press, Inc. AU rights of reproduction in any form reserved.
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ET AL.
lone on human peripheral blood leukocytes. A number of studies now suggest that the human immune response is regulated by functionally distinct subsets of T lymphocytes that can be distinguished by the presence of characteristic cell surface markers (13). The T-inducer/helper subset, which bears the T4 marker, facilitates the activation of other cells including T lymphocytes, B lymphocytes, macrophages, and hematopoietic stem cells. Suppressor T lymphocytes, which most often bear the T8 marker, modulate either the activity of the T-inducer/helper population or that of effector populations. The outcome of an immune response reflects the balance of inducer and suppressor cells which are specifically programmed to interact with antigen. Administration of agents such as cyclophosphamide or prednisolone might affect one or more subsets preferentially. Consequently, we have studied the effect of these agents upon the phenotype of human peripheral blood leukocytes, considering in particular the ratio of T4 + and T8 +cells. METHODS Patients and volunteers. All studies were conducted using protocols approved by the Human Subjects Protection Committee of the Sidney Farber Cancer Institute. Cyclophosphamide was administered to three men and two women with malignant melanoma. The patients’ ages ranged from 34 to 54 years with a median age of 39 years. Four of the five had undergone surgical resection of recurrent disease from lymph nodes or subcutaneous tissue. A fifth patient (J.R.) had undergone primary surgical resection of a superficial spreading melanoma from the right lower extremity. The lesion extended to Clarke’s level IV and measured 2.5 mm in greatest thickness. All patients were considered to be a greater than 50% risk of further disease recurrence. One patient (H.K.) had received previous adjuvant chemoimmunotherapy with dimethyltriazenoimidazolecarboxamide in combination with Bacillus Calmette-Guerin at another institution. Chemoimmunotherapy had been discontinued 80 days prior to the initiation of cyclophosphamide treatment. The remaining subjects had received only surgical treatment for their disease. On entry into the study, all patients were free from metastatic tumors that could be detected by physical examination, liver function studies, chest roentgenogram, cranial CT scan, or radionuclide scans of the liver and bone. During the trial no episodes of intercurrent infection were encountered and no medications other than cyclophosphamide were administered. Prednisolone was administered to six healthy male physician volunteers who ranged in age from 30 to 37 years with a median age of 34 years. Volunteers were chosen, rather than cancer patients, as we suspected that prednisolone might reduce, rather than increase the T4/T8 ratios. Participation was precluded by history of hypertension, diabetes, peptic ulcer disease, or granulomatous pulmonary disease. All subjects were normotensive and had normal chest roentgenograms. Administration of drugs. Cyclophosphamide (Cytoxan, Mead-Johnson, Evansville, Ind.) was administered intravenously over 30 min in 250 ml 5% dextrosenormal saline (Travenol, Deerfield, Ill.). The drug was given every 3 weeks and the dose was escalated from 50 to 700 mg/m2 as noted in Figs. l-4. Prednisolone
DRUG-INDUCED
MODULATION
OF
LEUKOCYTE
PHENOTYPE
103
sodium phosphate (Hydeltrasol, Merck Sharp & Dohme, West Point, Pa.) was administered intravenously over 15 min in 25 ml normal saline (Travenol). Each volunteer received only a single dose of 10 or 40 mg prednisolone/m2 at 8 AM. Control studies were performed at similar times of the day in the same individuals several weeks after prednisolone administration. Mononuclear cell preparation. Peripheral blood was anticoagulated with heparin sodium (Panheparin, Abbott Laboratories, North Chicago, Ill.). Leukocytes were enumerated with a flow cytometer (Coulter 5-plus, Coulter Electronics, Hialeah, Fla.). Smears were stained with Wright’s Giesma and differential counts were performed. Mononuclear cells were separated on Ficoll-Hypaque (Pharmacia Fine Chemicals, Division Pharmacia, Inc., Piscataway, N.J.) density gradients and cryopreserved as previously described (14). Cryopreservation under these conditions does not effect the phenotype of human peripheral blood mononuclear cells when compared before freezing and after thawing. Monoclonal antibodies. A series of monoclonal antibodies were used to define cell surface antigens on human lymphocytes and monocytes. Their production and characterization have been described in detail (13, 15). In brief, anti-T3 reacts with all peripheral T cells and approximately 10% of normal thymocytes (15). Anti-T4 and anti-T8 react with subpopulations of T cells which comprise 60% and 20-30% of peripheral T lymphocytes, respectively (16-18). The former population contains the human inducer subset (T4), whereas the latter antibody defines the cytotoxic/suppressor subset (T8). Ml reacts with peripheral blood monocytes, a small fraction of null cells, and with granulocytes that might contaminate the mononuclear cell preparation (19). Bl defines all peripheral blood B cells as well as 35% of lymph node lymphocytes (20). The anti-Ia monoclonal antibody defines a bimolecular glycoprotein antigen complex of 29,000- and 34,000-Da subunits. This complex is associated with resting B cells, a fraction of monocytes, and activated T cells, but not with resting T cells (15). Analysis of lymphocyte populations. After thawing, l-2 x lo6 lymphocytes were incubated at 4°C for 30 min in a 1: 250 dilution of one or the other monoclonal antibody and then washed twice in Eagle’s minimum essential medium supplemented with 5% fetal bovine serum. The cells were subsequently reacted with a 1: 40 dilution of goat anti-mouse IgG (Meloy Laboratories, Springfield, Va.), washed, and analyzed by flow cytometry on an Ortho cytofluorograph (FC 200/48OOA, Ortho Instruments, Westwood, Mass.) as previously described (21). Background staining was obtained by substituting 0.15 ml of a 1:250 dilution of control ascites from a mouse immunized with a nonproducing hybridoma clone. Absolute levels of cells bearing a given phenotype were calculated from the product of (a) leukocyte counts, (b) the percentage of mononuclear cells on differential count, and (c) the proportion of mononuclear cells which reacted with a particular monoclonal antibody. RESULTS
Cyclophosphamide was given intravenously to melanoma patients at 3-week intervals in gradually escalating doses of 50-700 mg/m2. Four patients completed at least three dose escalations. Tumor recurred in a fifth patient (H.K.) after he had
4.76 3.70 4.14 4.12 3.75 2.62
f k 2 + A r
1.03 0.14 0.13 1.34 1.48 1.18**
Granulocyte
rt 0.40 t 0.10*
1.00
0.70
0.44 0.03 0.13 0.12
k * t *
1.25 1.09 1.13 1.08
T3+
OBSERVED
AFTER
0.86 0.66 0.72 0.78 0.73 0.47
+ 0.42 + 0.15 +- 0.05 2 0.08 k 0.24 k 0.11***
T4+
ADMINISTRATION
1
0.52 0.38 0.38 0.27 0.20 0.25
f 0.20 f 0.05 k 0.13 f O.lO* 2 0.12** -+ 0.13**
TS+
Level”
OF DIFFERENT
TABLE DOSES
0.27 0.23 0.17 0.09 0.19 0.06
f 0.17 f 0.01 +- 0.10 -c o.ols* 2 0.22 I 0.02***
Ml+
OF CYCLOPHOSPHAMIDE
0.15 0.06 0.07 0.03 0.07 0.01
z? 0.04 k 0.09 2 0.07* 2 0.04* -t 0.09 + 0.01”
Bl+
f
0.07*
0.24 k 0.25 0.04 +- 0.02*
0.10
0.30 +- 0.15 0.15 f 0.11 0.18 k 0.14
Ia+
’ Mean c SD for the minimal value of each subset during biweekly monitoring of 2-5 patients. On the average, nadirs for cells bearing each of the phenotypic markers occurred 12.2- 13.7 days after administration of cyclophosphamide. Values are expressed x IO-$/liter of peripheral blood. Significance of difference from pretreatment values indicated by asterisks (paired t test, two tailed). * P < 0.05. **P < 0.025. *** 0.10 > P > 0.05.
600
400
0 50 100 200
mg/m2
Dose
NADIRS
F
in 4
$ 2
DRUG-INDUCED
MODULATION
OF
LEUKOCYTE
PHENOTYPE
105
received 100 mg cyclophosphamide/m2, requiring his removal from the study. Clinically, administration of the drug in these low doses was well tolerated. Only one patient developed mild nausea and vomiting at the highest dose level. Peripheral blood was obtained twice weekly to enumerate circulating leukocytes, to perform conventional differential counts and to determine the surface phenotype of mononuclear cells using monoclonal reagents. Granulocyte levels were minimally affected by single doses of cyclophosphamide ranging up to 400 mg/m2. A significant nadir was observed in each of four patients at 600 mg/m2 (Table 1). Smaller doses of the drug had more marked effects on different mononuclear cell populations. Modulation of subset levels was, however, transient. Values averaged over the 3 weeks between drug treatments did not differ significantly from control values. Examination of the lowest value attained during each course was more informative. B cells appeared particularly sensitive to cyclophosphamide with a significant decrease in the average nadir observed after 100 mg/m2 (Table 1). As the dose was escalated to 200 mg/m2, a decrease in T8+, Ml+, and Ia+ cells was also observed. Depression of T8+ cells was maintained at 400 and 600 mg/m2, although the latter dose of cyclophosphamide proved toxic for a variety of cell ‘types. T4+ cells proved resistant to cyclophosphamide in doses up to 400 mg/m2. Maintenance of T4+ levels and depression of T8+ levels produced transient elevations of the T4/T8 ratio in three of four patients (Figs. l-4). The T4/T8 ratio was evaluated at a fixed time of day for each of the four patients on 22-27 occasions. For each patient it was assumed that the difference between each serial observation and baseline observation was independently and normally distributed with constant variance. In this model, the chance of observing even one experimental value that exceeded three standard deviations from baseline ranged between 3 and 5% under the null hypothesis that cyclophosphamide would not affect the T4/T8 ratio over time. Patient-specific standard deviations used in these calculations were obtained from repeated measurements in the absence of cyclophosphamide and were quite similar to standard deviations observed in serial studies of healthy controls. An average standard deviation of 0.34 (range 0.280.40) was obtained for the TUT8 ratios on 2-4 occasions in each of four patients studied prior to treatment. A similar average standard deviation of 0.34 (range 0.23-0.45) was obtained for the T4/T8 ratios on 7-20 occasions in each of four healthy controls studied over 9-24 weeks. For three of the four patients who received cyclophosphamide, a difference in excess of three standard deviations was observed (P < 0.001). Elevations of the T4/T8 ratio were transient and appeared to depend critically upon dose. As the dosage of cyclophosphamide was escalated to 600 or 700 mg/m2, both T4+ and T8+ populations were reduced, decreasing the T4/T8 ratio. Prednisolone exerted a different effect upon the T4/T8 ratio of healthy male physician volunteers who had received either 40 or 10 mg/m2 of the corticosteroid intravenously on a single occasion. Within 3-6 hr after injection of 40 mg/m2, there was a marked decrease in the T4/T8 ratio which returned to normal within 24-48 hr (Fig. 5). When the same individuals were studied on different occasions without receiving prednisolone, a diurnal variation was observed in the T4/T8
BAST ET AL.
106
t 5.2 ,
4.8 I
.e----.
3.2 2.8 2.4
J Time (Weeks) FIG. l-4. Ratio of T4+JTS+ cells in the peripheral blood of four different melanoma patients at different times after the intravenous administration of cyclophosphamide (50-700 m&m*). The average standard deviation for two to four pretreatment determinations of T4flX in the patients studied was 0.34. Dashed lines indicate the pretreatment mean k 3 SD for each individual.
ratio which differed dramatically from the pattern observed following drug administration. In the absence of exogenous corticosteroids, the T4/T8 ratio increased during the course of the day (Fig. 5). Interestingly, injection of an intermediate dose of 10 mg prednisolone/m2 into three different volunteers produced an intermediate pattern (Fig. 5).
2.8
if 2 l-
2.4
-----------_--.
__-------------
2.0
1.6
1.2
Time (Weeks) FIGURE 2
4.2
3.8
if ;; I-
3.4
___-------..--------------
3.0 I-
2.:
l.i
I
I
I
I
I1
I
I
Time
11 (Weeks)
FIGURE 107
3
11
1 ”
108
BAST
ET AL.
2.4
t 2 2 I-
2.0/L 1.6
/
1.2 1 : 0.81
-----------------------------14’. Time FIGURE
(Weeks) 4
For statistical analysis, the T4/T8 ratios 6 hr after the administration of 40 mg prednisolone/m2 were compared to T4/T8 ratios obtained 6 hr after the initiation of the control study. Each subject was used as his own control and it was assumed that the difference between each serial observation and each baseline observation was normally distributed with a constant variance for both the control and prednisolone-treated groups. At 6 hr the differences between control and prednisolone groups were - 1.06, -2.49, and - 1.29. Under the assumptions that the standard deviation was 0.48 for each difference from baseline, and that the relative differences for control and prednisolone were independent, the 6-hr differences were statistically significant (P < 0.01) using a z test under the null hypothesis that prednisolone would fail to affect the T4/T8 ratio over time. When the percentages of different mononuclear cells were measured after administration of 40 mg prednisolone/m2 (Table 2) only the T4+ cells were affected selectively. However, when the levels of T4 and T8 were measured after administration of 40 mg prednisolone/m2 (Table 3) or 10 mg prednisolone/m2 (Table 4) it is apparent that both subsets were affected by the corticosteroid, but that the effect on T4+ cells was more marked than that on T8+ cells. Suppression of T4+ cells is observed up to 10 hr following 40 mg/m” (Table 3), but only up to 6 hr following 10 mg/m2 (Table 4). Administration of prednisolone affected not only T4+ and T8+ cells, but also reduced T3+, Ml+, Bl+, and Ia+ cells during the general lymphopenia that occurred within hours after injection. Levels of each of these cell types returned to normal within 24-48 hr and there was no selective decrease in the percentage of cells bearing these markers.
1.0
2.0
0
24 48
0 TIME
(HOURS)
24
1 Omg/m*
48
0
24
CONTROL
-
1 40
FIG. 5. Ratio of T4+/T8+ cells in the peripheral blood of normal volunteers at different intervals after the intravenous administration of 40 or 10 mg prednisolone/m*, or no prednisolone. Three of the four controls are the same individuals who received 40 mg prednisolone/m*.
c” G I-
3.0
40mg/ma
r
BAST ET AL.
110
TABLE PHENOTYPE
T3+
T4+
0
57.1 2 11.0
3 6
48.2 44.9 52.3 64.6 59.0
10
AFTERTHE
ADMINISTRATION
Percentage of mononuclear cells”
Interval (hr)
24 48
2
CELLSATDIFFERENTTIMES OF FWEDNI~~L~NE (40 mg/m2)
OFMONONUCLEAR
2 + lr k 2
6.5 4.1 1.4 2.4 5.3
44.0 30.8 23.8 28.7 45.1 39.6
2 2 k lr k +
1.2 0.4** 4.8* 4.8** 6.0 5.7
T8+ 23.7 26.5 26.8 27.2 23.3 22.6
k k k +-t -t
Ml+ 8.0 10.2 9.9 12.3 7.0 5.0
11.0 + 7.7 16.1 -+ 15.1 15.3 2 15.6 19.5 +- 11.0 11.3 2 3.5
11.1 2 4.4
Bl’ 5.0 4.0 5.7 5.4 4.8 4.0
k + t t 2 2
la+ 4.0 1.4 2.1 2.5 1.4 0.2
11.0 * 3.1 8.3 12.2 8.9 12.6 9.8
+ -+ i k -f
4.1 5.8 0.4 3.2 3.7
a Mean 2 SD for 3 individuals studied on the same day. Significance of difference from the 0 interval indicated by asterisks (paired t test, two tailed). * P < 0.05.
** P < 0.025.
DISCUSSION
The impact of cyclophosphamide on Bl+ peripheral blood lymphocytes from melanoma patients is consistent with earlier observations in animal systems where the drug exerted a greater effect on B cells than on T cells (l-4). Previous clinical studies have demonstrated that daily administration of cyclophosphamide in low doses can produce a persistent lymphopenia with a greater percentage decrease in E-rosette-negative cells than in E-rosette-positive cells (23). Treatment with cyclophosphamide at an average dose of 2.6 mg/kg can also suppress the humoral response to flagellin (24). Low doses of cyclophosphamide affected TV lymphocytes more markedly than T4+ cells. Suppressor activity has been associated with T8+ cells in a number of systems involving T-T and T-B interactions (17, 25). Additional studies will be required to determine whether the increases in the T4/T8 ratio induced with cyclophosphamide in viva are associated with augmented inducer and decreased suppressor functions. Cyclophosphamide-sensitive suppressor cells have been described in animals (7, 26-33) and in humans (34, 35). Precursors of suppressors are considered to be particularly susceptible to the cytotoxic effects of cyclophosphamide, whereas differentiated suppressors, inducers, and cytotoxic effecters are relatively more resistant (30-32). Some authors have proposed that cyclophosphamide-sensitive suppressor cells can be found among the T4+ population (35). Whether or not this proves to be the case, selective depletion of the TV population in viva may provide a useful marker for identifying doses of cyclophosphamide that affect the most sensitive immunoregulatory cells within the peripheral blood. Additional studies will be required to document that similar changes are achieved in other lymphoid compartments. Although it was possible to increase the T4/T8 ratio in three of four subjects, the effect was transient and critically dose dependent. Peaks in the T4/T8 ratio were related to the nadirs in levels of T8+ cells that occurred following the intermittent
x 10~Yliter
2.79 2 0.57 2.48 k 0.70
24 48
f SD, expressed
0.52 2 0.20 0.65 f 0.12 0.85 2 0.24
3 6 10
a Mean
2.10 -+ 0.77
Mononuclear cells
peripheral
1.80 zt 0.34 1.47 + 0.46
0.24 + 0.06 0.29 f 0.07 0.45 + 0.18
blood,
CELLS
1.20 + 0.69
T3+
OF MONONUCLEAR
0
Interval (hr)
PHENOTYPE
Ml+
0.32 0.26
0.10 0.09 0.17 + 0.13 f 0.05
f 0.12 k 0.07 + 0.11
Bl+
(40 mg/m2)
0.13 0.10
0.02 0.04 0.05
+ 0.03 + 0.03
+ 0.00 f 0.02 f 0.04
0.10 + 0.08
OF PREDNISOLONE
0.21 rf: 0.10
on the same day.
0.67 f 0.28 0.58 k 0.28
0.14 + 0.09 0.17 + 0.07 0.24 f 0.14
0.51 2 0.31
T8+
Level”
3 AFTER ADMINISTRATION
studied
TABLE TIMES
for 3 individuals
1.24 k 0.08 0.97 2 0.22
0.16 k 0.06 0.16 k 0.04 0.24 + 0.04
0.92 L 0.31
T4+
AT DIFFERENT
0.35 + 0.11 0.15 2 0.12
0.04 +- 0.01 0.08 f 0.05 0.07 2 0.02
0.25 + 0.14
Ia+
2 3
Ez 0
z
3 m
s 8
s
%
!
Is 5
BAST ET AL.
112
TABLE PHENOTYPE
Level”
Interval (hr) 0 3 6
10 24 48
4
OF MONONUCLEARCELLSATDIFFERENTTIMESAFTERADMINISTRATION OFPREDNI~~LONE (lOmg/mz)
0.95 0.28 0.31 0.74 1.13 1.24
T3+
T4+
T8+
Ml+
k k zk k ? 2
0.75 k 0.34 0.23 t 0.08 0.28 2 0.11 0.67 t 0.32
0.66 +- 0.37 0.28 + 0.08
0.29 -t 0.18 0.05 t 0.04
0.28 t 0.10
0.05 -e 0.04
0.52 2 0.28
0.23 + 0.12
1.09 k 0.39
0.71 _f 0.36
1.21 r 0.28
0.88 k 0.33
0.19 + 0.04 0.25 -t- 0.01
0.36 0.12 0.09 0.38 0.57 0.41
a Mean ? SD, expressed x lo-Vliter peripheral blood,
131’ 0.12
-t 0.02
Ia' 0.41 -e 0.07
0.04 2 0.01 0.04 -+-0.01 0.11 -+ 0.04
0.12 + 0.02 0.12 c 0.03 0.32 + 0.02
0.09
0.31 t 0.09
-t_ 0.05
0.12 t 0.04
0.35 2 0.03
for 3 individuals studied on the same day.
injection of cyclophosphamide at 3-week intervals. Daily administration of the drug in smaller doses might produce a more consistent effect on the T4/T8 ratio and facilitate studies of lymphocyte function. Our present observations do, however, suggest that cyclophosphamide can affect the T4/T8 ratio in cancer patients, and that further investigation is warranted. Modulation of suppressor cell function may be one requirement for effective immunotherapy of human malignancy. Cyclophosphamide has augmented both active and adoptive immunotherapy in murine systems (8). Whether cyclophosphamide will modulate the T4/T8 ratio in other disease states remains to be determined. The T8+ subset contains not only suppressors, but also cytotoxic T effecters capable of destroying allogeneic targets. Depletion of T8+ cells might result in a loss of cytotoxic T cells as well as cells with suppressor function. At present, however, it is not known whether T8+ cells are responsible for lysis of autologous tumor cells in cancer patients. In animal systems differentiated effecters are relatively resistant to low doses of cyclophosphamide. Cyclophosphamide at a sufficiently high dosage can inhibit both the primary and the anamnestic response to syngeneic transplantable murine tumor (9). Intravenous injection of prednisolone decreases, rather than increases, the T4/T8 ratio. Both T4+ and T8+ cells were affected, but there was a more marked depletion of the former. In guinea pigs, the administration of glucocorticoids has produced a redistribution of long-lived lymphocytes from the vascular compartment and into the bone marrow and spleen (36). A similar redistribution is likely to occur in man (10). Previous clinical studies have demonstrated a preferential depletion of T cells following glucocorticoid administration, with a greater effect on levels of T, cells than on levels of TG cells. Analysis of the TM and TG populations in an earlier report suggested that the TM population contained both T4+ and T8+ cells, whereas the TG population was depleted of T cells and enriched for Ia-negative cells which bore a monocyte-associated antigen (37). In the present study the effect of prednisolone on Ml cells was less marked than that on T4 and T8 cells, although levels of all mononuclear cells were decreased transiently. The phenotype of human peripheral blood leukocytes returned to a normal range within 24-
DRUG-INDUCED
MODULATION
OF LEUKOCYTE
PHENOTYPE
113
48 hr. As these studies were conducted in healthy volunteers, it remains to be seen whether glucocorticoids would have a similar effect upon the lymphoreticular subsets of patients with different diseases. A marked and consistent variation was observed in the T4/T8 ratio at different times of the day in the absence of prednisolone treatment. Whether or not this variation relates to endogenous glucocorticoid levels remains to be determined. Diurnal variation in the T4/T8 ratio had not been documented in the past. If useful data regarding T-cell subsets are to be obtained in future studies it is clear that peripheral blood must be obtained at similar times of the day. ACKNOWLEDGMENTS The authors wish to thank Heather Lane, Aggie Brennan, and Woon C. Yee for valuable technical assistance. Ms. Helen Thurman has provided excellent secretarial assistance.
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29. 30. 31. 32. 33. 34. 35.
Shand, F. L., and Liew, F. U., Eur. J. Immunol. 10, 480, 1980. Kaufmann, S. H. E., Hahn, H., and Diamentstein, T., J. Zmmunol. 125, 1104, 1980. Diamentstein, T., Klos, M., Hahn, H., and Kaufmann, S. H. E., J. Zmmunol. 126, 1717, 1981. Yu, S., Lannin, D. R., Tsiu-Collins, A. L., and McKhann, C. F., Cancer Res. 40, 2756, 1980. Hen@, J., Mokyr, M. B., Dray, S., Can&r Res. 40, 2135, 1980. Taube, D., Brown, Z., and Wiiiams, D. G.. Lnncet 1, 235, 1981. Ozer, H., Cowens, J. W., Colvin, M., Nussbaum-Blumenson, A., and Sheedy. D., J. Exp. Med. 155, 276, 1982. 36. Fauci, A. S., Immunology 28, 669, 1975. 37. Reinherz, E. L., Moretta, L., Roper, M., Breard, J. M., Mingari, M. C.. Copper, J. D., and Schlossman, S. F., J. Exp. Med. 151, 969, 1980. Received October 21, 1982; accepted with revisions January 10, 1983.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
28, 101-114 (1983)
Contrasting Effects of Cyclophosphamide on the Phenotype of Human Peripheral ROBERT Sidney
C. BAST,
Farber
Cancer
and Prednisolone Blood Leukocytes
JR., ELLIS L. REINHERZ, CAROLE AND STUART F. SCHLOSSMAN Institute,
Brigham Boston,
and Women’s Massachusetts
Hospital, 02115
MAVER, and Harvard
PHILIP Medical
LAVIN, School,
The cell surface phenotype of human peripheral blood mononuclear cells has been characterized before and after intravenous injection of cyclophosphamide or prednisolone. Low doses of cyclophosphamide (100-600 mg/m*) temporarily decrease levels of circulating B lymphocytes. Slightly higher doses of cyclophosphamide (200-600 mg/m”) produce transient depression of TS-, Ml-, and Ia-positive cells. After doses of 200400 mg cyclophosphamide/mz, TCpositive cells are spared, resulting in a transient elevation of the T4fT8 ratio. With higher doses of cyclophosphamide (2 600 mg/m”), all T cells are affected and the T4/T8 ratio declines to pretreatment levels. By contrast, intravenous injection of prednisolone at 40 mg/m* reduces the T4/T8 ratio. Levels of both T4 and TS cells decline, but T4 cells are affected more markedly than TS cells.
INTRODUCTION
The immunosuppressive effects of cyclophosphamide have been documented in animal systems and in clinical studies (1, 2). Inhibition of antibody synthesis has been achieved more readily than inhibition of delayed hypersensitivity after administration of the alkylator. In earlier reports both B cells and T cells decrease in human peripheral blood after cyclophosphamide injection. Selective depletion of B cells has, however, been observed in animal systems (3,4) where low doses of cyclophosphamide have potentiated delayed cutaneous reactivity to protein antigens (5) and contact allergens (6). Pretreatment with cyclophosphamide has eliminated tumor-specific suppressors in murine systems (7) and potentiated the therapeutic effect of immune T lymphocytes (8). At higher dosages, however, both the primary and the anamnestic response to tumor growth can be inhibited (9). Administration of glucocorticoids affects both the distribution and function of human lymphocytes (10). Maximal lymphopenia is observed 4-6 hr after steroid injections and lymphocyte levels return to the normal range in 24-48 hr. T cells are said to be affected more than B cells. Most studies have, however, measured E-rosette-forming cells and it is possible that glucocorticoids have affected expression of erythrocyte receptors rather than the actual number of circulating T cells. When T-cell subsets have been measured after injection of prednisolone, TM cells have been affected more markedly than TG cells in vivo (11). When studied in vitro, only the suppressor function attributed to TG cells has been inhibited (12).
The availability of monoclonal reagents which recognize subsets of T cells has permitted more precise analysis of the impact of cyclophosphamide and predniso101 0090-1229/83 $1.50 Copyrkht 0 1983 by Academic Press, Inc. AU rights of reproduction in any form reserved.
102
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lone on human peripheral blood leukocytes. A number of studies now suggest that the human immune response is regulated by functionally distinct subsets of T lymphocytes that can be distinguished by the presence of characteristic cell surface markers (13). The T-inducer/helper subset, which bears the T4 marker, facilitates the activation of other cells including T lymphocytes, B lymphocytes, macrophages, and hematopoietic stem cells. Suppressor T lymphocytes, which most often bear the T8 marker, modulate either the activity of the T-inducer/helper population or that of effector populations. The outcome of an immune response reflects the balance of inducer and suppressor cells which are specifically programmed to interact with antigen. Administration of agents such as cyclophosphamide or prednisolone might affect one or more subsets preferentially. Consequently, we have studied the effect of these agents upon the phenotype of human peripheral blood leukocytes, considering in particular the ratio of T4 + and T8 +cells. METHODS Patients and volunteers. All studies were conducted using protocols approved by the Human Subjects Protection Committee of the Sidney Farber Cancer Institute. Cyclophosphamide was administered to three men and two women with malignant melanoma. The patients’ ages ranged from 34 to 54 years with a median age of 39 years. Four of the five had undergone surgical resection of recurrent disease from lymph nodes or subcutaneous tissue. A fifth patient (J.R.) had undergone primary surgical resection of a superficial spreading melanoma from the right lower extremity. The lesion extended to Clarke’s level IV and measured 2.5 mm in greatest thickness. All patients were considered to be a greater than 50% risk of further disease recurrence. One patient (H.K.) had received previous adjuvant chemoimmunotherapy with dimethyltriazenoimidazolecarboxamide in combination with Bacillus Calmette-Guerin at another institution. Chemoimmunotherapy had been discontinued 80 days prior to the initiation of cyclophosphamide treatment. The remaining subjects had received only surgical treatment for their disease. On entry into the study, all patients were free from metastatic tumors that could be detected by physical examination, liver function studies, chest roentgenogram, cranial CT scan, or radionuclide scans of the liver and bone. During the trial no episodes of intercurrent infection were encountered and no medications other than cyclophosphamide were administered. Prednisolone was administered to six healthy male physician volunteers who ranged in age from 30 to 37 years with a median age of 34 years. Volunteers were chosen, rather than cancer patients, as we suspected that prednisolone might reduce, rather than increase the T4/T8 ratios. Participation was precluded by history of hypertension, diabetes, peptic ulcer disease, or granulomatous pulmonary disease. All subjects were normotensive and had normal chest roentgenograms. Administration of drugs. Cyclophosphamide (Cytoxan, Mead-Johnson, Evansville, Ind.) was administered intravenously over 30 min in 250 ml 5% dextrosenormal saline (Travenol, Deerfield, Ill.). The drug was given every 3 weeks and the dose was escalated from 50 to 700 mg/m2 as noted in Figs. l-4. Prednisolone
DRUG-INDUCED
MODULATION
OF
LEUKOCYTE
PHENOTYPE
103
sodium phosphate (Hydeltrasol, Merck Sharp & Dohme, West Point, Pa.) was administered intravenously over 15 min in 25 ml normal saline (Travenol). Each volunteer received only a single dose of 10 or 40 mg prednisolone/m2 at 8 AM. Control studies were performed at similar times of the day in the same individuals several weeks after prednisolone administration. Mononuclear cell preparation. Peripheral blood was anticoagulated with heparin sodium (Panheparin, Abbott Laboratories, North Chicago, Ill.). Leukocytes were enumerated with a flow cytometer (Coulter 5-plus, Coulter Electronics, Hialeah, Fla.). Smears were stained with Wright’s Giesma and differential counts were performed. Mononuclear cells were separated on Ficoll-Hypaque (Pharmacia Fine Chemicals, Division Pharmacia, Inc., Piscataway, N.J.) density gradients and cryopreserved as previously described (14). Cryopreservation under these conditions does not effect the phenotype of human peripheral blood mononuclear cells when compared before freezing and after thawing. Monoclonal antibodies. A series of monoclonal antibodies were used to define cell surface antigens on human lymphocytes and monocytes. Their production and characterization have been described in detail (13, 15). In brief, anti-T3 reacts with all peripheral T cells and approximately 10% of normal thymocytes (15). Anti-T4 and anti-T8 react with subpopulations of T cells which comprise 60% and 20-30% of peripheral T lymphocytes, respectively (16-18). The former population contains the human inducer subset (T4), whereas the latter antibody defines the cytotoxic/suppressor subset (T8). Ml reacts with peripheral blood monocytes, a small fraction of null cells, and with granulocytes that might contaminate the mononuclear cell preparation (19). Bl defines all peripheral blood B cells as well as 35% of lymph node lymphocytes (20). The anti-Ia monoclonal antibody defines a bimolecular glycoprotein antigen complex of 29,000- and 34,000-Da subunits. This complex is associated with resting B cells, a fraction of monocytes, and activated T cells, but not with resting T cells (15). Analysis of lymphocyte populations. After thawing, l-2 x lo6 lymphocytes were incubated at 4°C for 30 min in a 1: 250 dilution of one or the other monoclonal antibody and then washed twice in Eagle’s minimum essential medium supplemented with 5% fetal bovine serum. The cells were subsequently reacted with a 1: 40 dilution of goat anti-mouse IgG (Meloy Laboratories, Springfield, Va.), washed, and analyzed by flow cytometry on an Ortho cytofluorograph (FC 200/48OOA, Ortho Instruments, Westwood, Mass.) as previously described (21). Background staining was obtained by substituting 0.15 ml of a 1:250 dilution of control ascites from a mouse immunized with a nonproducing hybridoma clone. Absolute levels of cells bearing a given phenotype were calculated from the product of (a) leukocyte counts, (b) the percentage of mononuclear cells on differential count, and (c) the proportion of mononuclear cells which reacted with a particular monoclonal antibody. RESULTS
Cyclophosphamide was given intravenously to melanoma patients at 3-week intervals in gradually escalating doses of 50-700 mg/m2. Four patients completed at least three dose escalations. Tumor recurred in a fifth patient (H.K.) after he had
4.76 3.70 4.14 4.12 3.75 2.62
f k 2 + A r
1.03 0.14 0.13 1.34 1.48 1.18**
Granulocyte
rt 0.40 t 0.10*
1.00
0.70
0.44 0.03 0.13 0.12
k * t *
1.25 1.09 1.13 1.08
T3+
OBSERVED
AFTER
0.86 0.66 0.72 0.78 0.73 0.47
+ 0.42 + 0.15 +- 0.05 2 0.08 k 0.24 k 0.11***
T4+
ADMINISTRATION
1
0.52 0.38 0.38 0.27 0.20 0.25
f 0.20 f 0.05 k 0.13 f O.lO* 2 0.12** -+ 0.13**
TS+
Level”
OF DIFFERENT
TABLE DOSES
0.27 0.23 0.17 0.09 0.19 0.06
f 0.17 f 0.01 +- 0.10 -c o.ols* 2 0.22 I 0.02***
Ml+
OF CYCLOPHOSPHAMIDE
0.15 0.06 0.07 0.03 0.07 0.01
z? 0.04 k 0.09 2 0.07* 2 0.04* -t 0.09 + 0.01”
Bl+
f
0.07*
0.24 k 0.25 0.04 +- 0.02*
0.10
0.30 +- 0.15 0.15 f 0.11 0.18 k 0.14
Ia+
’ Mean c SD for the minimal value of each subset during biweekly monitoring of 2-5 patients. On the average, nadirs for cells bearing each of the phenotypic markers occurred 12.2- 13.7 days after administration of cyclophosphamide. Values are expressed x IO-$/liter of peripheral blood. Significance of difference from pretreatment values indicated by asterisks (paired t test, two tailed). * P < 0.05. **P < 0.025. *** 0.10 > P > 0.05.
600
400
0 50 100 200
mg/m2
Dose
NADIRS
F
in 4
$ 2
DRUG-INDUCED
MODULATION
OF
LEUKOCYTE
PHENOTYPE
105
received 100 mg cyclophosphamide/m2, requiring his removal from the study. Clinically, administration of the drug in these low doses was well tolerated. Only one patient developed mild nausea and vomiting at the highest dose level. Peripheral blood was obtained twice weekly to enumerate circulating leukocytes, to perform conventional differential counts and to determine the surface phenotype of mononuclear cells using monoclonal reagents. Granulocyte levels were minimally affected by single doses of cyclophosphamide ranging up to 400 mg/m2. A significant nadir was observed in each of four patients at 600 mg/m2 (Table 1). Smaller doses of the drug had more marked effects on different mononuclear cell populations. Modulation of subset levels was, however, transient. Values averaged over the 3 weeks between drug treatments did not differ significantly from control values. Examination of the lowest value attained during each course was more informative. B cells appeared particularly sensitive to cyclophosphamide with a significant decrease in the average nadir observed after 100 mg/m2 (Table 1). As the dose was escalated to 200 mg/m2, a decrease in T8+, Ml+, and Ia+ cells was also observed. Depression of T8+ cells was maintained at 400 and 600 mg/m2, although the latter dose of cyclophosphamide proved toxic for a variety of cell ‘types. T4+ cells proved resistant to cyclophosphamide in doses up to 400 mg/m2. Maintenance of T4+ levels and depression of T8+ levels produced transient elevations of the T4/T8 ratio in three of four patients (Figs. l-4). The T4/T8 ratio was evaluated at a fixed time of day for each of the four patients on 22-27 occasions. For each patient it was assumed that the difference between each serial observation and baseline observation was independently and normally distributed with constant variance. In this model, the chance of observing even one experimental value that exceeded three standard deviations from baseline ranged between 3 and 5% under the null hypothesis that cyclophosphamide would not affect the T4/T8 ratio over time. Patient-specific standard deviations used in these calculations were obtained from repeated measurements in the absence of cyclophosphamide and were quite similar to standard deviations observed in serial studies of healthy controls. An average standard deviation of 0.34 (range 0.280.40) was obtained for the TUT8 ratios on 2-4 occasions in each of four patients studied prior to treatment. A similar average standard deviation of 0.34 (range 0.23-0.45) was obtained for the T4/T8 ratios on 7-20 occasions in each of four healthy controls studied over 9-24 weeks. For three of the four patients who received cyclophosphamide, a difference in excess of three standard deviations was observed (P < 0.001). Elevations of the T4/T8 ratio were transient and appeared to depend critically upon dose. As the dosage of cyclophosphamide was escalated to 600 or 700 mg/m2, both T4+ and T8+ populations were reduced, decreasing the T4/T8 ratio. Prednisolone exerted a different effect upon the T4/T8 ratio of healthy male physician volunteers who had received either 40 or 10 mg/m2 of the corticosteroid intravenously on a single occasion. Within 3-6 hr after injection of 40 mg/m2, there was a marked decrease in the T4/T8 ratio which returned to normal within 24-48 hr (Fig. 5). When the same individuals were studied on different occasions without receiving prednisolone, a diurnal variation was observed in the T4/T8
BAST ET AL.
106
t 5.2 ,
4.8 I
.e----.
3.2 2.8 2.4
J Time (Weeks) FIG. l-4. Ratio of T4+JTS+ cells in the peripheral blood of four different melanoma patients at different times after the intravenous administration of cyclophosphamide (50-700 m&m*). The average standard deviation for two to four pretreatment determinations of T4flX in the patients studied was 0.34. Dashed lines indicate the pretreatment mean k 3 SD for each individual.
ratio which differed dramatically from the pattern observed following drug administration. In the absence of exogenous corticosteroids, the T4/T8 ratio increased during the course of the day (Fig. 5). Interestingly, injection of an intermediate dose of 10 mg prednisolone/m2 into three different volunteers produced an intermediate pattern (Fig. 5).
2.8
if 2 l-
2.4
-----------_--.
__-------------
2.0
1.6
1.2
Time (Weeks) FIGURE 2
4.2
3.8
if ;; I-
3.4
___-------..--------------
3.0 I-
2.:
l.i
I
I
I
I
I1
I
I
Time
11 (Weeks)
FIGURE 107
3
11
1 ”
108
BAST
ET AL.
2.4
t 2 2 I-
2.0/L 1.6
/
1.2 1 : 0.81
-----------------------------14’. Time FIGURE
(Weeks) 4
For statistical analysis, the T4/T8 ratios 6 hr after the administration of 40 mg prednisolone/m2 were compared to T4/T8 ratios obtained 6 hr after the initiation of the control study. Each subject was used as his own control and it was assumed that the difference between each serial observation and each baseline observation was normally distributed with a constant variance for both the control and prednisolone-treated groups. At 6 hr the differences between control and prednisolone groups were - 1.06, -2.49, and - 1.29. Under the assumptions that the standard deviation was 0.48 for each difference from baseline, and that the relative differences for control and prednisolone were independent, the 6-hr differences were statistically significant (P < 0.01) using a z test under the null hypothesis that prednisolone would fail to affect the T4/T8 ratio over time. When the percentages of different mononuclear cells were measured after administration of 40 mg prednisolone/m2 (Table 2) only the T4+ cells were affected selectively. However, when the levels of T4 and T8 were measured after administration of 40 mg prednisolone/m2 (Table 3) or 10 mg prednisolone/m2 (Table 4) it is apparent that both subsets were affected by the corticosteroid, but that the effect on T4+ cells was more marked than that on T8+ cells. Suppression of T4+ cells is observed up to 10 hr following 40 mg/m” (Table 3), but only up to 6 hr following 10 mg/m2 (Table 4). Administration of prednisolone affected not only T4+ and T8+ cells, but also reduced T3+, Ml+, Bl+, and Ia+ cells during the general lymphopenia that occurred within hours after injection. Levels of each of these cell types returned to normal within 24-48 hr and there was no selective decrease in the percentage of cells bearing these markers.
1.0
2.0
0
24 48
0 TIME
(HOURS)
24
1 Omg/m*
48
0
24
CONTROL
-
1 40
FIG. 5. Ratio of T4+/T8+ cells in the peripheral blood of normal volunteers at different intervals after the intravenous administration of 40 or 10 mg prednisolone/m*, or no prednisolone. Three of the four controls are the same individuals who received 40 mg prednisolone/m*.
c” G I-
3.0
40mg/ma
r
BAST ET AL.
110
TABLE PHENOTYPE
T3+
T4+
0
57.1 2 11.0
3 6
48.2 44.9 52.3 64.6 59.0
10
AFTERTHE
ADMINISTRATION
Percentage of mononuclear cells”
Interval (hr)
24 48
2
CELLSATDIFFERENTTIMES OF FWEDNI~~L~NE (40 mg/m2)
OFMONONUCLEAR
2 + lr k 2
6.5 4.1 1.4 2.4 5.3
44.0 30.8 23.8 28.7 45.1 39.6
2 2 k lr k +
1.2 0.4** 4.8* 4.8** 6.0 5.7
T8+ 23.7 26.5 26.8 27.2 23.3 22.6
k k k +-t -t
Ml+ 8.0 10.2 9.9 12.3 7.0 5.0
11.0 + 7.7 16.1 -+ 15.1 15.3 2 15.6 19.5 +- 11.0 11.3 2 3.5
11.1 2 4.4
Bl’ 5.0 4.0 5.7 5.4 4.8 4.0
k + t t 2 2
la+ 4.0 1.4 2.1 2.5 1.4 0.2
11.0 * 3.1 8.3 12.2 8.9 12.6 9.8
+ -+ i k -f
4.1 5.8 0.4 3.2 3.7
a Mean 2 SD for 3 individuals studied on the same day. Significance of difference from the 0 interval indicated by asterisks (paired t test, two tailed). * P < 0.05.
** P < 0.025.
DISCUSSION
The impact of cyclophosphamide on Bl+ peripheral blood lymphocytes from melanoma patients is consistent with earlier observations in animal systems where the drug exerted a greater effect on B cells than on T cells (l-4). Previous clinical studies have demonstrated that daily administration of cyclophosphamide in low doses can produce a persistent lymphopenia with a greater percentage decrease in E-rosette-negative cells than in E-rosette-positive cells (23). Treatment with cyclophosphamide at an average dose of 2.6 mg/kg can also suppress the humoral response to flagellin (24). Low doses of cyclophosphamide affected TV lymphocytes more markedly than T4+ cells. Suppressor activity has been associated with T8+ cells in a number of systems involving T-T and T-B interactions (17, 25). Additional studies will be required to determine whether the increases in the T4/T8 ratio induced with cyclophosphamide in viva are associated with augmented inducer and decreased suppressor functions. Cyclophosphamide-sensitive suppressor cells have been described in animals (7, 26-33) and in humans (34, 35). Precursors of suppressors are considered to be particularly susceptible to the cytotoxic effects of cyclophosphamide, whereas differentiated suppressors, inducers, and cytotoxic effecters are relatively more resistant (30-32). Some authors have proposed that cyclophosphamide-sensitive suppressor cells can be found among the T4+ population (35). Whether or not this proves to be the case, selective depletion of the TV population in viva may provide a useful marker for identifying doses of cyclophosphamide that affect the most sensitive immunoregulatory cells within the peripheral blood. Additional studies will be required to document that similar changes are achieved in other lymphoid compartments. Although it was possible to increase the T4/T8 ratio in three of four subjects, the effect was transient and critically dose dependent. Peaks in the T4/T8 ratio were related to the nadirs in levels of T8+ cells that occurred following the intermittent
x 10~Yliter
2.79 2 0.57 2.48 k 0.70
24 48
f SD, expressed
0.52 2 0.20 0.65 f 0.12 0.85 2 0.24
3 6 10
a Mean
2.10 -+ 0.77
Mononuclear cells
peripheral
1.80 zt 0.34 1.47 + 0.46
0.24 + 0.06 0.29 f 0.07 0.45 + 0.18
blood,
CELLS
1.20 + 0.69
T3+
OF MONONUCLEAR
0
Interval (hr)
PHENOTYPE
Ml+
0.32 0.26
0.10 0.09 0.17 + 0.13 f 0.05
f 0.12 k 0.07 + 0.11
Bl+
(40 mg/m2)
0.13 0.10
0.02 0.04 0.05
+ 0.03 + 0.03
+ 0.00 f 0.02 f 0.04
0.10 + 0.08
OF PREDNISOLONE
0.21 rf: 0.10
on the same day.
0.67 f 0.28 0.58 k 0.28
0.14 + 0.09 0.17 + 0.07 0.24 f 0.14
0.51 2 0.31
T8+
Level”
3 AFTER ADMINISTRATION
studied
TABLE TIMES
for 3 individuals
1.24 k 0.08 0.97 2 0.22
0.16 k 0.06 0.16 k 0.04 0.24 + 0.04
0.92 L 0.31
T4+
AT DIFFERENT
0.35 + 0.11 0.15 2 0.12
0.04 +- 0.01 0.08 f 0.05 0.07 2 0.02
0.25 + 0.14
Ia+
2 3
Ez 0
z
3 m
s 8
s
%
!
Is 5
BAST ET AL.
112
TABLE PHENOTYPE
Level”
Interval (hr) 0 3 6
10 24 48
4
OF MONONUCLEARCELLSATDIFFERENTTIMESAFTERADMINISTRATION OFPREDNI~~LONE (lOmg/mz)
0.95 0.28 0.31 0.74 1.13 1.24
T3+
T4+
T8+
Ml+
k k zk k ? 2
0.75 k 0.34 0.23 t 0.08 0.28 2 0.11 0.67 t 0.32
0.66 +- 0.37 0.28 + 0.08
0.29 -t 0.18 0.05 t 0.04
0.28 t 0.10
0.05 -e 0.04
0.52 2 0.28
0.23 + 0.12
1.09 k 0.39
0.71 _f 0.36
1.21 r 0.28
0.88 k 0.33
0.19 + 0.04 0.25 -t- 0.01
0.36 0.12 0.09 0.38 0.57 0.41
a Mean ? SD, expressed x lo-Vliter peripheral blood,
131’ 0.12
-t 0.02
Ia' 0.41 -e 0.07
0.04 2 0.01 0.04 -+-0.01 0.11 -+ 0.04
0.12 + 0.02 0.12 c 0.03 0.32 + 0.02
0.09
0.31 t 0.09
-t_ 0.05
0.12 t 0.04
0.35 2 0.03
for 3 individuals studied on the same day.
injection of cyclophosphamide at 3-week intervals. Daily administration of the drug in smaller doses might produce a more consistent effect on the T4/T8 ratio and facilitate studies of lymphocyte function. Our present observations do, however, suggest that cyclophosphamide can affect the T4/T8 ratio in cancer patients, and that further investigation is warranted. Modulation of suppressor cell function may be one requirement for effective immunotherapy of human malignancy. Cyclophosphamide has augmented both active and adoptive immunotherapy in murine systems (8). Whether cyclophosphamide will modulate the T4/T8 ratio in other disease states remains to be determined. The T8+ subset contains not only suppressors, but also cytotoxic T effecters capable of destroying allogeneic targets. Depletion of T8+ cells might result in a loss of cytotoxic T cells as well as cells with suppressor function. At present, however, it is not known whether T8+ cells are responsible for lysis of autologous tumor cells in cancer patients. In animal systems differentiated effecters are relatively resistant to low doses of cyclophosphamide. Cyclophosphamide at a sufficiently high dosage can inhibit both the primary and the anamnestic response to syngeneic transplantable murine tumor (9). Intravenous injection of prednisolone decreases, rather than increases, the T4/T8 ratio. Both T4+ and T8+ cells were affected, but there was a more marked depletion of the former. In guinea pigs, the administration of glucocorticoids has produced a redistribution of long-lived lymphocytes from the vascular compartment and into the bone marrow and spleen (36). A similar redistribution is likely to occur in man (10). Previous clinical studies have demonstrated a preferential depletion of T cells following glucocorticoid administration, with a greater effect on levels of T, cells than on levels of TG cells. Analysis of the TM and TG populations in an earlier report suggested that the TM population contained both T4+ and T8+ cells, whereas the TG population was depleted of T cells and enriched for Ia-negative cells which bore a monocyte-associated antigen (37). In the present study the effect of prednisolone on Ml cells was less marked than that on T4 and T8 cells, although levels of all mononuclear cells were decreased transiently. The phenotype of human peripheral blood leukocytes returned to a normal range within 24-
DRUG-INDUCED
MODULATION
OF LEUKOCYTE
PHENOTYPE
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48 hr. As these studies were conducted in healthy volunteers, it remains to be seen whether glucocorticoids would have a similar effect upon the lymphoreticular subsets of patients with different diseases. A marked and consistent variation was observed in the T4/T8 ratio at different times of the day in the absence of prednisolone treatment. Whether or not this variation relates to endogenous glucocorticoid levels remains to be determined. Diurnal variation in the T4/T8 ratio had not been documented in the past. If useful data regarding T-cell subsets are to be obtained in future studies it is clear that peripheral blood must be obtained at similar times of the day. ACKNOWLEDGMENTS The authors wish to thank Heather Lane, Aggie Brennan, and Woon C. Yee for valuable technical assistance. Ms. Helen Thurman has provided excellent secretarial assistance.
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