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[CANCER RESEARCH 47, 3317-3321, June 15, 1987] Effect of Low Dose Cyclophosphamide on the Immune System of Cancer Patients: Reduction of T-Suppressor Function without Depletion of the CD8+ Subset1 David Herd ' and Michael J. Mastrangelo Division of Oncology, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 ABSTRACT Low dose cyclophosphamide (CY) can augment the development of delayed-type hypersensitivity to primary antigens in patients with ad vanced cancer. In this paper, we have considered the hypothesis that the immunopotentiation is related to reduction of T-suppressor activity. Peripheral blood lymphocytes were collected and cryopreserved from 45 patients with metastatic malignancy before and then 3, 7, and 19 days after administration of CY, 300 mg/mz i.v. The peripheral blood lympho cytes were tested for generation of concanavalin A-inducible suppressor activity, proliferative response to phytohemagglutinin, and phenotype using monoclonal antibodies to ( '1)4 and CDS. Concanavalin A-inducible suppression was significantly reduced by day 3 and declined progressively through day 19. The mean percentage changes in suppression were: day 3, -23.4 ±6.8 (SE) (P < 0.01); day 7, -33.1 ±143 (P = 0.052); day 19, -43.1 ±10.7 (P < 0.01). In contrast, CY caused no significant changes in phytohemagglutinin proliferation (mean percentage changes: day 3, -4.7 ±6.1; day 7, -15.6 ±7.5; day 19, -5.5 ±8.1), indicating that the reduction in concanavalin A-inducible suppression was not merely a reflection of a general reduction in peripheral blood lymphocyte func tion. The total number of circulating lymphocytes was not affected by low dose CY. Moreover, flow cytometric analysis showed no significant changes in the percentage of circulating CD8+ or CD4+ T-cells or in the CD4/CD8 ratio at any time point after CY. Thus, administration of low dose CY to these patients caused impairment of nonspecific T-suppressor function without selective depletion of the CD8+ subset that is generally associated with that function. Several immunoregulatory models that are consistent with these observations are discussed. INTRODUCTION Cyclophosphamide is a potent immunosuppressive agent that can also augment a variety of immune responses (1). The major determinant of whether CY3 suppresses or potentiates immu nity is the time of its administration in relationship to the presentation of antigen. Thus, when given following antigen, CY can suppress the immune response but often augments the response when given 1-3 days prior to antigen (2). In experimental systems, CY has been shown to augment immunity by its selective toxicity to T-lymphocyte-mediated suppressor function (3). This finding is of more than theoretical interest, because reduction in T-suppression may be an essential component of successful immunotherapy, as indicated by sev eral murine tumor systems (4). For example, Berendt and North Received 1/2/87; revised 3/20/87; accepted 3/24/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by a grant from the National Cancer Institute (CA39248) and by funds from the Nat Pincus Endowment. 2To whom requests for reprints should be addressed, at Division of Medical Oncology, Thomas Jefferson University, 1015 Walnut St., Room 1005, Phila delphia, PA 19107. 3The abbreviations used are: CY, cyclophosphamide; PBL, peripheral blood lymphocytes; Con A, concanavalin A; PHA, phytohemagglutinin; 1)111. delayedtype hypersensitivity; MNC, mononuclear cells. (4) have shown that depletion of T-suppressor cells by CY markedly facilitates the eradication of an established tumor by adoptively transferred immune lymphocytes. For the past several years, we have been attempting to apply these principles to the treatment of human cancer. We observed that pretreatment of patients with CY, 300 mg/m2 i.v., 3 days before injection of antigen augmented the development of de layed-type hypersensitivity, both to keyhole limpet hemocyanin (6) and to autologous tumor cells (7). With this publication, we have begun to investigate the immunological basis of these findings. The data will show that administration of CY is followed by reduction in Con A-inducible T-suppressor function of peripheral blood lymphocytes without a corresponding de pletion of the suppressor-cytotoxic (CDS-)-) subset. MATERIALS AND METHODS Patients. The study population consisted of patients with surgically incurable, metastatic cancer. Initially, PBL from 13 patients were studied for Con A-inducible suppression, PHA stimulation, and surface phenotype. Subsequently, the surface phenotype studies were expanded by the addition of 32 patients to make a total of 45 study subjects. A summary of the clinical characteristics of these patients is shown in Table 1. Thirty-seven of the study subjects had malignant melanoma; of the other 8, 7 had colorectal carcinoma and 1 had breast adenocarcinoma. Despite the presence of advanced visceral métastasesin most of these patients, their performance status was good (median Karnovsky score, 80). Although 34 patients had been treated previously with chemotherapy and 7 had received radiation therapy, all such cytotoxic therapy had been discontinued for at least 4 weeks prior to entering this study. No patients were given corticosteroids while under study. Treatment. The patients for this study were drawn from two previ ously published studies: study 1, the effect of low dose CY on the immunological response to primary, non-tumor-related antigens (6) ( 15 patients); and study 2, the effect of low dose CY on the development of delayed-type hypersensitivity to autologous tumor cells (7) (30 pa tients). CY was given at a dose of 300 mg/m2 by rapid i.v. infusion. Three days after CY, the patients from study 1 were sensitized with either dinitrochlorobenzene or keyhole limpet hemocyanin, and the patients on study 2 were given i.d. injections of irradiated, autologous tumor cells mixed with Bacillus Calmette-Guérin.Heparinized blood was obtained from all patients just prior to CY infusion (day 0) and then 3, 7, and 19 days afterwards. No additional treatment was given during the study period. Preparation of PBL. As described previously (6), PBL were separated by density gradient centrifugation on Ficoll:metrizoate, suspended in freezing medium [consisting of KI'M I 1640 (Grand Island Biological Co., Grand Island, NY), penicillin, streptomycin, 10% pooled human AB+ serum (Hint-ell Laboratories, Carson, CA), and 10% dimethyl sulfoxide], frozen in a controlled rato freezer, and stored in liquid nitrogen. When needed for testing, MNC were thawed rapidly, diluted slowly to gradually reduce the concentration of dimethyl sulfoxide, and washed twice. Lymphocyte Phenotype Studies. The MNC were stained by incubât 3317 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research. LOW DOSE CYCLOPHOSPHAMIDE 1Tumor Table characteristics of the patients378291659 typeMelanomaOtherSexMenWomenAgeKarnovsky statusMétastasesVisceralSkin (27-79)'80 (40-100)36911142077 onlyPrioror nodal chemotherapies01>1Prior AND T-SUPPRESSOR FUNCTION calculated the change between the pre-CY sample and each post-CY sample. Then for a given parameter, we determined whether the mean change, or percentage change, of the whole group was significantly different from zero at each time point by using the Student t test for nonindependent variables (2 tailed). This approach avoids the potential problem of mistaking spontaneous fluctuations for CY-induced changes. RESULTS Con A-inducible Suppression. The generation of nonspecific suppressor T-cells was studied in 13 patients before and 3, 7, therapySurvival radiation and 19 days after low dose CY. Suppressor function was signif (1-50+) (mo)Clinical icantly reduced by day 3 and declined progressively through * Median (range). day 19. The mean values (percentage of suppression) were: day 0,42.4%; day 3, 33.4%; day 7, 30.4%; day 19, 26.8%. The data ing 0.5 x IO6 cells with one of the following antibodies on ice for 20 were analyzed by an independent t test (2 tailed) after calculat min: anti-Leu 3a (anti-CD4; helper-inducer T-cells) (8); anti-Leu 2a ing the percentage change in suppression at each time point (anti-CD8; suppressor-cytotoxic T-cells) (8); and anti-Leu M3 (monocompared with the pretreatment (day 0) value (Fig. 1). The cytes) (9); all were directly conjugated to fluorescein isothiocyanate mean percentage changes were: day 3, —23.4±6.8 (P < 0.01); (green fluorescence) or phycoerythrin (red fluorescence) (Becton-Dickday 7, -33.1 ±14.3 (P = 0.052); day 19, -43.1 ±10.7 (P < inson, Mountainview, CA). Negative fluorescence controls were ob 0.01). The percentage change in suppression on day 19 com tained by using fluorescein isothiocyanate-conjugated mouse IgG of pared with day 3 was also significant (mean percentage change, irrelevant specificity. -27.3 ±10.8, P < 0.025). A similar degree of impairment of Analysis of cell surface markers was performed with a Coulter EPICS Con A-inducible suppression was observed whether patients C flow cytometer (Coulter Electronics, Hialeah, FL). Lymphocytes were sensitized on day 3 with non-tumor antigens or with tumor were examined by setting bit map gates on a plot of forward versus 90degree light scatter, which excluded monocytes (10); absence of mon - cells (Fig. 1, *, O, respectively). ocvtes in the bit map was confirmed by lack of staining with anti-Leu PHA Proliferation. In parallel, PBL from this group of pa tients were tested for their proliférât ive response to an optimal M3. Blood leukocytes were enumerated in a standard manner with a concentration of PHA. In contrast to its effect on Con A Coulter Counter (Coulter Electronics), and differential counts were suppressors, CY caused no significant changes in PHA reactiv performed by examination of Giemsa-stained blood smears. Total ity. The mean responses (cpm) for the group were as follows: MNC was calculated by adding the proportions of lymphocytes and day 0, 101, 949; day 3, 98,439; day 7, 104,415; day 19, 89,415. monocytes, as determined by Giemsa smears, and multiplying the sum The percentage changes at each time point were analyzed as by the total blood leukocyte count. The lymphocyte count was deter described above: day 3, -4.7 ±6.1; day 7, -15.6 ±7.5; day 19, mined by subtracting monocytes [% Leu M3(+)/100 x total MNC] -5.5 ±8.1 (P > 0.05 for each) (Fig. 2). Similar results were from total MNC. obtained when a suboptimal concentration of PHA (0.1 /¿g/ml) Con A-inducible Suppression. This was performed by a modification was used (data not shown). Thus, the progressive reduction in of the method of Shou et al. (11), which has been described previously (6). MNC (2 x 10') were incubated in 1 ml of culture medium with or Con A-inducible suppression seen after CY was not merely a without Con A (25 ¿ig/ml)in plastic tubes in a 37"C, 5% CU2 incubator reflection of a general reduction in peripheral blood lymphocyte for 3 days. Then they were washed and exposed to mitomycin C, 25 function. ¿tg/ml(Sigma Chemical Co., St. Louis, MO), for 30 min in a 37°C Blood Lymphocyte Composition. Initially, PBL from 10 of water bath. After 3 washes, they were suspended in culture medium, and 5 x IO4cells were added to 1 x 10* "indicator" cells in microtiter wells. The latter were MNC from a normal donor, and the same donor V40.0-20.0-o.o.-20. was used in all the experiments described herein. Then PHA was added to all the wells at a predetermined suboptimal concentration (0.1 ¿¿g/ ml). The mixtures were incubated at 37°Cin 5% COj for 3 days and Per then pulsed with ['"Ijiododeoxyuridine (New England Nuclear, Boston, Cc MA) and harvested with an automatic harvestor. The percentage of he suppression was calculated as a 0--40.0-60.0-80.0.-100.0.B6M -p<.01*90M0il nt cpm (1 + UN) - cpm (1 + Con A) ga 100 x e cpm (I + UN) -P-.OKMBH0g -p<.018N gPBO. where cpm (I + UN) are the uptake of ['"Ijiododeoxyuridine by indicator cells in the presence of unstimulated patient's cells, and cpm 11 (I + Con A) are the uptake by indicator cells in the presence of Con Astimulated patient's cells. 19 Proliferativi.' Response to PHA. This was performed by a standard Days After Cyclophosphamide Fig. 1. Percentage changes in Con A-inducible suppression after cyclophostechnique, described previously (6). The concentration of PHA was 1.0 phamide. Each marker represents the percentage change, compared with day 0, (jg/ml. in Con A-inducible suppression generated from the PBL from a single patient. >, Analysis of Data. Lymphocyte functional assays were performed in immunized with non-tumor antigens; O, immunized with tumor cells. />, calcu triplicate and the mean was calculated. Replicate values never varied lated by a t test for nonindependent samples, has been placed adjacent to the by more than 20% For each patient and for each parameter, we mean. 3318 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research. LOW DOSE CYCLOPHOSPHAMIDE AND T-SUPPRESSOR FUNCTION 40.0-20.0-P Oj40.0h e. nnP "•"c C -p-NSN hSS-»-"'t • *if• M0M3Ä0•*•"ÕNIIMN7 20.0-an9 ga e -40.0 9e-60.0-80. ntagBCD60. nu. ft u*-M.O-++ !$ i1 1!! -|Hß$* ;A.t4+; 0K8Ng 19Days After -P-«-• -P-« *kt $ + CyclophosphamidePee• Fig. 2. Percentage changes in PHA stimulation after Cyclophosphamide. Each marker represents the percentage change, compared with day 0, in PHA stimu lation (cpm) of the PBL from a single patient. *, immunized with non-tumor antigens; O, immunized with tumor cells. P, calculated by a r test for nonindependent samples, has been placed adjacent to the mean. .V.V.not significant. -40.0 19 CD8+80.0-,60.0pe ++ the original 13 patients were studied. To reduce the possibility that small changes in lymphocyte composition would be missed because of inadequate sample size, PBL that had been obtained and cryopreserved from an additional 32 patients were studied, and the results were analyzed compositely. The number of data points at each time point was: day 0, 42; day 3, 40; day 7, 27; day 19, 12. The total number of circulating lymphocytes was not affected by a single administration of low dose CY; the values (number of cells per mm3) were: day 0, 1620 ±112; day 3, 1681 ±132; day 7, 1602 ±152; day 19,1576 ±135. Prior to CY, the T-cell composition (percentage) measured by flow cytometric analysis, was: CD4+ T-cells, 46.6 ±2.0; CD8+ T-cells, 19.4 ±1.3; CD4/ CDS ratio, 3.2 ±0.4. Fig. 3 shows the percentage change in circulating CD8+ and CD4+ T-cells for each of the study patients following administration of low dose CY. For a partic ular patient, there was sometimes considerable variation in the proportion of cells in each subset from one time point to another. However, analysis of the group demonstrated that administration of CY was not followed by any significant changes in circulating CD8+ or CD4+ T-cells or in the CD4/ CD8 ratio, despite a significant change in Con A suppression and, clinically, an augmented induction of DTH to keyhole limpet hemocyanin or autologous melanoma cells. DISCUSSION The original observation, some 20 years ago, that CY could potentiate immune responses (12) had been corroborated and extended by a number of investigators. Most studies have demonstrated augmentation of cell-mediated immunity, usually measured as delayed-type hypersensitivity, to an array of anti gens, varying from microbial products to syngeneic testicular tissue (13, 14). Under some circumstances antibody responses have also been heightened (15). CY-mediated immunopotentiation has been achieved in ex perimental animals with a remarkably wide range of dosages from 60 to 2100 mg/m2 (2, 16). However, both in vivo and in t. +*• 40.0r Cc 20.0,e h aÃŽS o.o,a e9 -ao.O-e-40. Ã-n +i * +• i * ' ^^G-pHC —TÃ-f^Jr1 ±J* J. t y T -^"«S +È +* *'f 0--60. i ÃŽ" 0-+ 3 7 19 DaysAfter Cyclophosphamide Fig. 3. Percentage changes in circulating CD4+ T-cells and CD8+ T-cells after Cyclophosphamide. Each marker represents the percentage change, com pared with day 0, for a single patient. /'. calculated by a / test for nonindependent samples, has been placed adjacent to the mean. .V.V.not significant. does not require enzymatic activation) at a concentration of 3 Mg/ml, while suppressor T-cells were inactivated at a concentra tion of 1 Mg/ml or less. Our laboratory was the first to demonstrate that CY could augment the human immune response in vivo (17). We showed that the administration of a conventional cytotoxic dose of CY, 1000 mg/m2, to patients with advanced cancer 3 days before sensitization with antigen resulted in augmentation of DTH to the primary antigen, keyhole limpet hemocyanin, with no effect on the antibody response to that antigen. In a subsequent study (6), we reported that a much lower dose of CY, 300 mg/m2, was just as effective in augmenting the acquisition of DTH and heightened the antibody response as well. More recently, we have shown that "low dose" CY potentiated the acquisition of DTH to autologous melanoma cells, which in two cases was followed by regression of metastatic tumor (7). In the present study, we have tested the hypothesis that reduction of nonspecific T-suppressor function might be at least partially responsible for the immunopotentiating effects of low dose CY. Using cryopreserved PBL from patients with ad vitro evidence suggests that lower concentrations of CY may be vanced cancer who had been treated with CY in one of our advantageous in that they are selectively toxic to the T-supprevious studies, we observed a striking impairment of Con A pressor system and sparing of other T-cell functions. Thus, inducible suppressor activity. This reduction was detectable by Kauffman et ai. (3) reported that T-cells capable of adoptively the third day after CY administration and increased progres transferring DTH were resistant to 4-hydroperoxy-CY (which sively through day 19. The reduction in suppressor function on 3319 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research. LOW DOSE CYCLOPHOSPHAMIDE day 3 is consistent with the irnmunopotentiation studies in both humans and experimental animals (2,6, 7). Thus, sensitization with antigen on day 3, a time when nonspecific suppressor function is low, might result in impaired generation of antigenspecific suppression as well, and thus more efficient production of T-cells that mediate DTH and provide help for antibody production. The persistence of the impairment of Con A sup pression through day 19 is more difficult to correlate, since in the few human studies and in most of the published animal experiments the interval between CY and immunization has been no more than 3 days. However, at least one laboratory (18) has shown that immunopotentiation can be obtained in mice when antigen is given as long as 7 days after CY, and in humans the potentiation window may be considerably wider. Since Con A-inducible suppression is generally associated with the CD8+ subset of T-cells (19), it is noteworthy that the impairment ofl -suppressor activity was associated neither with a fall in the percentage of circulating CD8+ cells nor with a change in the so-called "helper/suppressor" ratio, i.e., %CD4+/ %CD8+. Previously, we found that high dose (1000 mg/m2) CY inhibited Con A-inducible suppression without changing the relative proportions of CD4+ and CD8+ T-cells, but in that study, there was a marked reduction in the total blood lymphocyte concentration and thus a fall in the absolute number of CD8+ T-cells (20). In contrast, in the current study low dose CY reduced suppressor function without affecting either the relative or the absolute numbers of this subset. These results are in conflict with data published by Bast et al. (21) who reported that low doses of CY (200-400 mg/m2) did produce selective depletion of circulating CD8+ T-lymphocytes in patients with melanoma. However, their conclusion was based on the study of only 4 patients, 3 of whom exhibited a significant change. Moreover, they administered multiple, escalating doses of CY to each patient and thus could have been measuring a cumulative effect rather than the effect of a single dose. We believe that our study (testing a single administration of CY, using a much larger sample size, and performing statis tical analysis by group) provides a more accurate assessment of the effects of low dose CY on peripheral blood T-cell compo sition. There are several immunoregulatory models that are consist ent with the observation that CY reduces Con A-inducible suppression without depleting CD8+ T-cells. Ozer et al. (22) have shown that in vitro exposure of human lymphocytes to 4hydroperoxy-CY prevents the generation of suppressor T-cells by Con A but does not inhibit the suppressor cells after they have been induced. They postulated the existence of a minor, extremely CY-sensitive, population of "presuppressor" cells. Secondly, Damle et al. (23) have studied a T-cell with the phenotype CD4+, Leu 8+, which is not a suppressor but is necessary for the induction of CD8+ suppressors. Finally, evidence has accumulated that CD4+ T-cell themselves can become potent suppressors after stimulation with Con A (24). Morimoto et al. (25) have suggested such cells bear the newly recognized surface molecule, 2H4. Partial depletion of, or sublethal damage to, one of these subpopulations of CDS- lym phocytes by CY could be reflected in reduction of T-suppressor activity and, consequently, in augmentation of immune re sponses. AND T-SUPPRESSOR FUNCTION ACKNOWLEDGMENTS The authors wish to thank the following individuals for their inval uable assistance in the completion of this project: Ellen Hart, R.N., who performed the patient-related work; Marsha Golden, who per formed the flow cytometry; and Cannella Clark, who performed the lymphocyte functional assays. REFERENCES 1. Herd, I). Maguire, H. C., Jr., and Mastrangelo, M. J. Immunopotentiation by cyclophosphamide and other cytotoxic agents. In: R. Feniche) and M. Chingos (eds.). Immune Modulating Agents, pp. 39-61. New York: Marcel Dekker, 1985. 2. Turk, J. I... and Parker, D. Effect of cyclophosphamide on immunological control mechanisms. Immunol. Rev., 65: 99-113, 1982. 3. Kaufmann, S. H. E., Hahn, H., and Diamantstein, T. Relative susceptibilities of T cell subsets involved in delayed-type hypersensitivity to sheep red blood cells to the in vitro action of 4-hydroperoxycyclophosphamide. J. Immunol., 125:1104-1108, 1980. 4. Berendt, M. J., and North, R. 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The role of the 2H4 molecule in the generation of suppressor function in Con A-activated T cells. J. Immunol., 137: 32473253, 1986. 3321 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1987 American Association for Cancer Research. Effect of Low Dose Cyclophosphamide on the Immune System of Cancer Patients: Reduction of T-Suppressor Function without Depletion of the CD8+ Subset David Berd and Michael J. Mastrangelo Cancer Res 1987;47:3317-3321. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/47/12/3317 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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