ICANCER RESEARCH 50. 1160-1 164. Februar} 15. 1990) Enhancement by 4 of - or Antibody-induced Proliferation of Lymphocytes from Interleukin 2-treated Cancer Patients1

Jonathan Treisman, Carl M. Higuchi, John A. Thompson, Steven Gillis, Catherine G. Lindgren, Donald E. Kern, Stanley R. Ridell, Philip D. Greenberg, and Alexander I efer Department of Medicine, Division of Medical Oncolog); University of Washington School of Medicine, Seattle. Has/tinglan 9X195 fJ. T., C. M. H.,J. A. T., C, G. L., D. E. K., S. R. R., P. D. G., A. F.]; and Immunex Corporation, Seattle, H'ashington 98101 [S. G.J

ABSTRACT IL-2-activated cells for therapy are usually generated by ob taining PBMC from cancer patients shortly after systemic IL- Systemic interleukin 2 (IL-2) and IL-2-activated lymphocytes have 2 therapy and culturing them with IL-2 in vitro. The cells thus induced tumor regression in some cancer patients. The IL-2-activated cells have usually been generated by obtaining peripheral blood mono- generated for infusion are phenotypically and functionally het nuclear cells (PB1V1C)from cancer patients shortly after systemic IL-2 erogeneous and contain cells with LAK activity (5, 7), as well therapy and culturing them with IL-2 in vitro. In an effort to augment as other cells which may mediate or contribute to antitumor the ex vivogeneration of such cells preactivated in vivo, we examined the responses (8). In an effort to enhance the generation of such proliferative responses of PBIVIC from IL-2-treated cancer patients to cells i/i vitro, several agents which induce a proliferative re several proliferative signals including IL-2, interleukin 4 (II -4). and sponse in normal human PBMC were studied for their possible mitogenic antibodies to CD3 and CD28. Although much is known about proliferative effects on PBMC from IL-2-treated patients. the response of normal PBMC to these signals, the possibility was IL-4 is a multipotent which enhances prolifera considered that the response of lymphocytes preactivated by IL-2 in vivo tion of B-cells, T-cells, natural killer cells, and (9- might differ from that of normal PBMC. Accordingly, PBIVICobtained 17). In normal lymphocytes, IL-4 is comitogenic with antigen from ten normal, healthy controls and from 17 patients with advanced cancer 1 to 3 days after systemic IL-2 therapy were cultured for 4 days (15), lectins (16, 17), and antibodies to CD3 (17). IL-4 can with 11.-4(1000 units/ml) and/or IL-2 (10 units/ml or 1000 units/ml) or augment the generation of antigen-specific cytotoxic T-cells with combinations of 11.-I and ant ¡-('1)3±anti-CD28, and they were (18-20) and can induce non-major histocompatibility complex- then tested for proliferation by ('Hjthymidine incorporation. IL-4 failed restricted LAK activity in human PBMC preactivated with IL- to induce proliferation of normal PBMC and inhibited IL-2-induced 2 (21, 22). In contrast to the pleotropic effects of IL-4, mono proliferation, whereas IL-4 alone induced proliferation in PBMC from clonal antibodies directed against CD3 (anti-CD3) specifically five of 11 IL-2-treated patients and did not inhibit but augmented the activate and induce the proliferation of CD3+ T-cells (23) and proliferation induced by IL-2 (10 units/ml and 1000 units/ml) in PBMC can support the growth of major histocompatibility complex- from six of nine patients and five of 11 patients, respectively. Anti-CD3 restricted T-cells (24) as well as cells with LAK activity (25). induced proliferation in PBMC from eight of nine patients, and the The proliferative response to anti-CD3 can be enhanced using proliferation was consistently augmented by coculture with anti-CD28. Finally, IL-4 significantly augmented the proliferative responses of monoclonal antibodies directed against CD28, a M, 44,000 PBMC from IL-2-treated patients to anti-CD3, as well as to the combi homodimer found on approximately 80% of peripheral T-cells nation of anti-CD3 and anti-CD28. Thus, in PBMC from IL-2-treated (26). The proliferative response of normal lymphocytes to anti- cancer patients, IL-4 enhanced the in vitro proliferation induced by IL-2 CD3 is also known to be enhanced by IL-4. or by anti-CD3 ±anti-CD28. The results suggest that IL-4 and/or Although much is known about the proliferative response of mitogenic antibodies may be useful in augmenting the ex vivo generation normal lymphocytes to these agents, the response of lympho of lymphocytes for clinical adoptive ¡mmunotherapy. cytes from IL-2-treated patients, i.e., cells which have been preactivated in vivo by IL-2, and, potentially, by other inter INTRODUCTION mediary and , may be different from that of normal lymphocytes. For example, IL-4 inhibits IL-2-in- The systemic administration of IL-2'1 and autologous lym duced LAK activity in normal human PBMC, but enhances IL- phocytes activated by culture with IL-2 has induced tumor 2-induced LAK activity in PBMC preactivated by IL-2 (21, 22). regression in some cancer patients (1-3). However, the re Accordingly, the proliferative effects of IL-4 and mitogenic sponses have been observed in only a minority of patients, and antibodies on PBMC from IL-2-treated cancer patients were the doses of IL-2 required have usually been toxic (4, 5). In examined. The data demonstrate that (a) in contrast to the animal models, the antitumor effects observed correlated with inhibition of IL-2-induced proliferation by IL-4 in normal the amount of IL-2 administered and with the number of PBMC, IL-4 alone induced substantial proliferation and aug activated cells infused (6). Thus, the development of techniques mented the proliferative response to IL-2 in PBMC from the to augment the generation of activated lymphocytes ex vivo majority of the patients tested; (¿>)anti-CD3induced prolifera may potentially enhance tumor responses in vivo. One approach tion of patient PBMC; (c) anti-CD28 enhanced the proliferative to this goal is the use of other proliferative signals in addition response to anti-CD3; and (d) IL-4 augmented the proliferative to or in lieu of IL-2. response to both mitogenic antibodies. The results suggest that Received 10/10/89: accepted 11/13/89. IL-4 ±mitogenic antibodies may have a role in the ex vivo The costs of publication of this article were defrayed in part by the payment generation of lymphocytes for adoptive trials. 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. ' This work was supported in part by Grant CA 09515 and Contract NO1- CM47668 awarded by the National Cancer Institute. Department of Health and MATERIALS AND METHODS Human Services. Some of the data in this manuscript were presented at the 80th Annual Meeting of the American Association for Cancer Research. May 1989. Study Population and Blood Sampling. Seventeen patients aged 30 to ' To whom requests for reprints should be addressed, at the Department of 71 yr with metastatic renal cell carcinoma (9 patients), melanoma (6 Medicine. Division of Oncology, RM-17, University of Washington, Seattle. WA patients), diffuse histiocytic lymphoma (one patient), and carcinoma of 98195. JThe abbreviations used are: IL-2, interleukin 2; IL-4, interleukin 4; LAK, the colon (one patient) participated in a Phase I/II trial of IL-2 and lymphokine-activated killer; PBMC, peripheral blood mononuclear cells; dThd, adoptive transfer of LAK cells (3, 4). Patients had no preceding treat thymidine; p55, M, 55,000 protein; TIL, tumor-infiltrating lymphocytes. ments within 4 wk of study and had Karnofsky scores >70% at initiation 1160 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. IL-4 ENHANCEMENT OF PROLIFERATION AFTER IL-2 THERAPY of IL-2 therapy. All patients received systemic recombinant human of IL-2, which is normally used for LAK cell generation in IL-2 (Hoffman-LaRoche Inc., Nutley, NJ), with treatment cycles con clinical trials (1-3), was also studied. The results are shown in sisting of IL-2 administered by continuous i.v. infusion for 5 consecutive Table 1. IL-4 alone failed to induce proliferation and inhibited days. Seven patients received 1 to 4 treatment cycles of IL-2 (3 x IO6 IL-2-driven proliferation in PBMC from healthy controls (n = units/m2/day) (4, 5), and 10 patients received a single cycle of IL-2 at a higher dose (6 x IO6units/m2/day) (3). Blood samples were collected 7), as previously reported (16, 22, 31). By contrast, IL-4 induced proliferation in PBMC from 5 of 11 IL-2-treated patients. 1 to 3 days after termination of a treatment cycle, which corresponded Furthermore, rather than inhibit proliferation induced by IL-2, to the time of initial leukapheresis for ex vivo cell culture for LAK generation. Blood was collected from healthy controls on the day of IL-4 augmented proliferation induced by IL-2 (10 units/ml or assay. 1000 units/ml) in PBMC from 6 of 9 and 5 of 11 IL-2-treated Preparation of Human Blood Mononuclear Cells and T-Cells. PBMC patients, respectively. were isolated from heparinized peripheral blood by Ficoll-Hypaque Dependence of the Proliferative Effects of IL-4 upon Preacti density gradient separation. For purification of T-cells, PBMC were vation of Lymphocytes by IL-2 in Vivo or in Vitro. To determine adherence depleted of followed by resetting with 2-amino- whether the ability of IL-4 to augment IL-2-induced prolifera ethyl-isothiouronium bromide-treated sheep RBC (27). The resultant tion in PBMC from IL-2-treated cancer patients reflected the population was >90% CD3* and <0.5% Mo3+ as assessed by fluores effects of systemically administered IL-2 or the presence of cence-activated cell sorting. Lymphokines. Purified recombinant human IL-4 was kindly provided cancer, PBMC were obtained from a patient with metastatic by Immunex Corp., Seattle, WA, with a specific activity of IO4 units/ melanoma before and after IL-2 therapy and cultured in IL-4 Mgas determined in a human B-cell proliferation assay (18). Human (1000 units/ml) ±IL-2 (1000 units/ml). The results are pre recombinant IL-2 was kindly provided by Hoffman-LaRoche and had sented in Table 2. As in normal PBMC, IL-4 inhibited IL-2- a specific activity of IO6units/^g as measured in a cytotoxic T-lympho- induced proliferation of patient PBMC before IL-2 therapy, cyte line (CTLL) proliferation assay (28). whereas in PBMC obtained 24 h after therapy, IL-4 did not Monoclonal Antibodies. Murine monoclonal antibodies 64.1 (IgG2a inhibit, but rather augmented IL-2-induced proliferation. Thus, anti-CD3) and 9.3 (IgG2a anti-CD28) have been described previously the altered proliferative response of the PBMC to IL-4 appeared (26). Antibodies purified from ascites fluid by Protein A-Sepharose to depend upon systemic therapy with IL-2. affinity chromatography were kindly provided by Dr. Paul Martin, Fred The effects of systemic IL-2 may be secondary to the secretion Hutchinson Cancer Research Center, Seattle, WA. Lymphocyte Proliferation Assays. Cells were cultured at 37°Cin of intermediary lymphokines or cytokines in vivo. To determine whether the proliferative effects of IL-4 on PBMC preactivated complete medium consisting of RPMI 1640 supplemented with peni cillin, streptomycin, L-glutamine, and heat-inactivated human AB with IL-2 i/i vitro would be similar to those of cells preactivated serum (15%). In the proliferation assays, PBMC or purified T-cells i/i vivo, PBMC from normal healthy individuals were preincu- were cultured in triplicate for 4 days at 2.5 x IO5cells/well in 96-well bated with IL-2 (250 units/ml) for 4 days and then tested for a flat-bottomed culture plates with various combinations of IL-2, IL-4, proliferative response to IL-4, IL-2, and to a combination of anti-CD3, and anti-CD28. Anti-CD3 and anti-CD28 were used in IL-2 and IL-4. The results, presented in Table 3, demonstrate soluble form except with purified T-cells, in which anti-CD3 was that IL-4 induced proliferation and augmented the proliferation immobilized onto the wells of flat-bottomed plates (29). Six to 8 h induced by IL-2 (10 units/ml) in cells preincubated with IL-2 prior to harvesting, 1 ¿iCiof [3H]dThd (6.7 Ci/mM specific activity; but not in fresh PBMC. New England Nuclear Corp., Boston, MA) was added to cultures. Cells were harvested onto glass fiber filters, and [3H]dThd incorporation was Anti-CD3 Induction of Proliferation of PBMC from IL-2- treated Patients and Anti-CD28 Enhancement of This Response. quantified by liquid scintillation counting. Criteria used to define pro liferation or the enhancement of proliferation were (experimental cpm Anti-CD3 is mitogenic in normal PBMC (23), and anti-CD28 •¿+•controlcpm) > 1.5 and (experimental cpm - control cpm) > 500 augments that response (26). However, systemic IL-2 has a cpm. variety of immunomodulatory effects, and the response of lym In the experiments involving in vitro IL-2 preactivation, PBMC were phocytes from IL-2-treated patients to mitogenic signals may cultured with IL-2 (250 units/ml) at 2.5 x IO6 cells/ml in 24-well differ from that of normal lymphocytes (32). To determine the culture plates. After 4 days, the cells were harvested, washed extensively proliferative response to anti-CD3 (l Mg/ml) ±anti-CD28 (2 with phosphate-buffered saline, and then further cultured in prolifera Mg/ml) of PBMC obtained from patients after IL-2 therapy, tion assays as described above. PBMC obtained from patients after IL-2 therapy and from healthy controls were cultured for 4 days with anti-CD3 ±anti- RESULTS CD28 and tested for proliferation. The results are shown in Table 4. Anti-CD3 induced proliferation in both PBMC from Augmentation by IL-4 of IL-2-driven Proliferation of PBMC 6 of 6 normal controls in 13 of 13 experiments and in PBMC from IL-2-treated Patients. To determine the proliferative re from 8 of 9 IL-2-treated patients in 8 of 9 experiments. How sponse to IL-4 of PBMC preactivated by the systemic admin ever, the responses to anti-CD3 of PBMC from patients (me istration of IL-2 in vivo, PBMC were obtained from patients 1 dian cpm = 2,700) were significantly lower than that by PBMC to 3 days after IL-2 therapy, were cultured for 4 days with IL- from controls (median cpm = 16,399, P < 0.05 by rank-sums 4 (1000 units/ml) alone or with a combination of IL-4 (1000 test). Anti-CD28 augmented the proliferative response to anti- units/ml) plus IL-2 (10 units/ml or 1000 units/ml), and were CD3 in PBMC from 4 of 4 healthy controls and in PBMC then tested for [3H]dThd incorporation. The time for obtaining from 5 of 5 IL-2-treated patients. PBMC from patients was selected on the basis of the observa Comitogenic Effect of IL-4 and Anti-CD3 ±Anti-CD28 upon tion that the 1 to 3 days after completing IL-2 therapy repre PBMC from IL-2-treated Patients. IL-4 is a potent costimulator sents the time of maximal rebound lymphocytosis and expres of proliferation when used in combination with or sion of activation markers on PBMC (5, 30), and the time when anti-CD3 in normal PBMC (14-17). To determine its effect on patient PBMC are usually obtained by leukapheresis for ex vivo PBMC preactivated by IL2, PBMC from IL-2-treated patients LAK cell generation for infusion. The lower concentration of were cultured with combinations of IL-4 (1000 units/ml), anti- IL-2 which induces suboptimal proliferation was used to facil CD3 (1 Mg/ml), and anti-CD28 (2 ng/m\) and tested for prolif itate detection of enhancing or inhibitory effects of IL-4. The eration. The results are shown in Table 5. IL-4 significantly effect of IL-4 on proliferation induced by the high concentration and, in some cases, dramatically augmented anti-CD3-induced 1161 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. IL-4 ENHANCEMENT OF PROLIFERATION AFTER IL-2 THERAPY

Table 1 IL-4 augmentation of IL-2-induced proliferation of PBMC from IL-2-treated patients PBMC from healthy controls or IL-2-treated patients were cultured for 4 days with IL-2 (10 units/ml or 1.000 units/ml) and/or IL-4 (1000 units/ml) and tested for proliferation. cpm MediumHealthy units/ml)No10 units/ml)No(1000 IL-4526 IL-4614 IL-47,816 IL-41.503 IL-415,709 1L-49,881 7)PatientsR.controls (n = ±86°702230135394330218152232207185200+±1003,5211,8857234591932686163587551701,070lL-2(±2,5748032.4394421,0834862538575735,876NT*NT+±2799.9413.6131,3572,3897157357,26099513,743NTNTIL-2±3,4036,23122.31010.70114.39311,3627.87778,17878.72751,02229,2899,781+±2,22616.66031,51517,12115,23017,2458,11467,71739,67543,28542.68525,747

K.J. B.L. G.R. H.CO.H.

M.H.S.R.

R.B.C.W.

T.S.S."

Mean ±SEM.* NT, not tested.No

Table 2 Proliferative effects of IL-4 altered as a result of IL-2 therapy Table 4 Anti-CDi induction of proliferation of PBMC from IL-2-treated patients PBMC »ereobtained from a patient immediately before and 1 day after IL-2 and anti-CD2S augmentation of this response therapy cultured with IL-2 (1000 units/ml) ±IL-4 (1000 units/ml) for 4 days, PBMC obtained from healthy controls or from IL-2-treated patients were and tested for proliferation. IL-4 inhibited IL-2-induced proliferation of PBMC cultured with anti-CD3 ±anti-CD28 for 4 days and then tested for proliferation. obtained before therapy. In contrast, IL-4 did not inhibit IL-2-induced prolifera controlsS. Healthy patientsMedium702380177200185485579639287cpmAnti-CD39.8262,7008,0531,4843891,0686,86114,8991,371Anti-CD3 tion of PBMC obtained after therapy, but rather augmented proliferation mildly. cpm Medium IL-4 IL-2 IL-2 + IL-4 +anti-CD285.5087,67093,24441,382NT*NTNTNTNTNTNTNTNTR.+anti-CD2822,5499,65917,33412,2701,704NTNTNTNT Before IL-2 B.°J. After IL-2249 18514935716,765 29,2899,352 42,685 K.J. T.D. N.J. T.K. W.S.S.W. Table 3 IL-4 induction of proliferation and augmentation of the proliferative K.°A. response of normal PBMC preac t¡votedwith IL-2 in vitro R.S. T.J. B.°K. H.J. cpm N.S. B.O.P.D. B.°K. +IL-44,860 K.°K. M.IL-2-treated Experiment1Experiment K.°K. PreactivatedFresh112,2553.267214.9133,737155,323IL-249,624125,21133.641203.29310.815 K.°K. K."D. 2Cells"Fresh T."Medium430727789195243383130427167225385219904cpmAnti-CD31,1811,59853.40116,20421,03536,94388.40314,54767,5148,3042,01538,25210.815Anti-CD3 PreactivatedMedium5,43026,863IL-41,460 111,352IL-2 172,548 " Indicates an individual tested more than once. " PBMC from healthy volunteers were cultured for 4 days with IL-2 (250 * NT, not tested. units/ml) and then tested for a proliferative resposne to IL-4, IL-2 (10 units/ml), or to a combination of IL-2 and IL-4. Their response was compared with that of Table 5 IL-4 enhancement of the proliferative response of PBMC from cells tested fresh, without preactivation by IL-2. IL-2-treated patients to anti-CD} ±anti-CD2S PBMC obtained from IL-2-treated patients were cultured with combinations proliferation of PBMC from 7 of 8 patients. IL-4 further of anti-CD3. anti-CD28, and IL-4 (1000 units/ml) for 4 days and then tested for augmented the proliferative responses to the combination of proliferation. anti-CD3 and anti-CD28 in PBMC from 5 of 6 patients tested. Induction of Proliferation by IL-4 and Anti-CD3 of T-Cells +anti-CD28 from IL-2-treated Patients. Although anti-CD3 specifically +anti-CD284.8171,939NTNTNTNT6,54417,33412,2701.704Anti-CD3+IL-431,37413,058NTNTNTNT9,65938,82924,0353,077 IL-4NTNT13,90220.82173,85139,0583,30522,58910,6391,357cpmAnti-CD3 stimulates the proliferation of T-cells, IL-4 can affect a variety PatientRK-2L. of cell lineages, including macrophages (12) and non-T-cells G.D.M.J. (9-11, 13). Therefore the mechanism by which IL-4 augments the proliferative response of PBMC from IL-2-treated patients H.O.P.J. may include effects on accessory cells, which are important in B.J. anti-CD3-induced proliferation (23). To characterize the cells N.J. proliferating in response to IL-4 and anti-CD3, the proliferative W.S.S.W. response and accessory cell dependence of purified T-cells from T.Anti-CD3NT-NT1,3711,06814,8996,8612,7008,0531,484389Anti-CD3H- IL-2-treated patients in response to IL-4 and anti-CD3 were •¿NT,not tested. examined. T-cells were purified by erythrocyte rosetting (27), cultured with IL-4 (1000 units/ml) and anti-CD3 (1 Mg/ml or significantly augmented proliferation without a requirement for immobilized) for 4 days, and then tested for proliferation. The accessory cells. Thus, IL-4 and anti-CD3 induced proliferation results are shown in Table 6. As reported with normal T-cells of purified T-cells from PBMC preactivated by IL-2 in vivo, (23), T-cells preactivated by IL-2 in vivo did not proliferate in and the effect of IL-4 was not strictly accessory cell dependent. response to soluble anti-CD3, but the addition of plastic-adher ent accessory cells at the beginning of culture restored the DISCUSSION response. By contrast, T-cells exhibited proliferation in re A potential way to improve the efficacy of systemic IL-2 sponse to anti-CD3 immobilized onto plastic dishes, and IL-4 therapy of human cancer is to develop techniques to augment 1162 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. lL-4 ENHANCEMENT OF PROLIFERATION AFTER II.-2 THERAPY

Table 6 Proliferation of T-cellsfrom lL-2-treated patients in response to IL-4 ative response observed might be a function of either decreased and anti-CD3 Purified T-cells or PBMC from an IL-2-treated patient were cultured for 4 lymphokine production or decreased lymphokine receptor days with IL-4 (250 units/ml) and/or anti-CD3 (1 fig/ml) and tested for prolif expression. No evidence for either hypothesis has yet been eration. obtained. Although IL-4 induces little or no proliferation in normal, anti- resting PBMC (16), IL-4 augments the proliferation of T-cells CD3No ami-CD3No costimulated by antigen (15), (16, 17), or antibodies to the CD3 molecular complex (17). Similarly, IL-4 enhanced With With IL-4IL-48,053 IL-4IL-432,951 the proliferation of PBMC from IL-2-treated patients costim PBMCPurified 22,589 54,267 ulated with anti-CD3. The enhancement of proliferation of T-cells 214223cpmSoluble376 3,219 42,202 58,703 normal PBMC by IL-4 appears to be mediated by both IL-2- Purified T-cells + AC°Medium177 6,131 18,160Immobilized25.643 39,402 dependent and IL-2-independent pathways. Antibodies to the °AC, adherent cells added to purified T-cells to serve as accessory cells. p55 IL-2 receptor (anti-Tac) inhibit augmentation of anti-CD3- induced proliferation by IL-4 (17), suggesting that costimula- the ex vivo generation of activated lymphocytes for adoptive tion by IL-4 may increase autocrine IL-2 production. However, transfer (6). Lymphocytes for therapy have been generated by anti-Tac does not completely block the augmentation, and IL- culturing PBMC obtained from cancer patients shortly after 4 augments the proliferation of T-cells in the presence of systemic IL-2 therapy with IL-2 in vitro. IL-2 administration cyclosporin A which blocks endogenous IL-2 production (17), produces a rebound lymphocytosis (5, 30), which allows for a suggesting that IL-4 can act in an IL-2-independent manner. higher recovery of cells for ex vivo culture. Systemic IL-2 also Our observation that IL-4 could augment the proliferation of activates PBMC in vivo, with the increased expression of acti IL-2-treated PBMC induced by an optimal concentration of IL- vation markers HLA-DR and TaC (p55 IL-2 receptor) on 2 (1000 units/ml) is consistent with the hypothesis that IL-4 PBMC (5, 30), and the induction of circulating LAK effector does more than increase endogenous IL-2 production. IL-4 can cells (5). In an effort to augment the ex vivo generation of augment the expression of the p55 IL-2 receptor induced by activated cells, we have examined the proliferative response of low concentrations of IL-2 (1 unit/ml) in human PBMC preac PBMC from IL-2-treated patients, i.e., cells preactivated by IL- tivated by IL-2 in vitro (22). Thus, the enhancement of anti- 2 in vivo, to IL-4, anti-CD3, and anti-CD28. IL-4 inhibits the CD3-induced proliferation by IL-4 of PBMC preactivated by proliferative response of normal PBMC to IL-2, but augments IL-2 in vivo may reflect an upregulation of IL-2 receptor expres the proliferation of lymphocytes preactivated by IL-2 in vitro sion. (22). Therefore, it was hypothesized that IL-4 might augment CD28 is a M, 44,000 cell surface homodimer present on IL-2-induced proliferation in PBMC from IL-2-treated pa approximately 80% of T-cells (26). The natural ligand for CD28 tients, i.e., PBMC preactivated with IL-2 in vivo. The results is not known, but monoclonal antibodies to CD28, although demonstrate that, in PBMC from IL-2-treated patients, IL-4 not mitogenic alone, can function as second signals to induce induces proliferation and augments IL-2-driven proliferation. increased IL-2 production, IL-2 receptor expression, and pro In addition, anti-CD3 induced proliferation of PBMC from IL- liferation of T-cells which have been primarily activated by anti- 2-treated patients which proliferation was enhanced by anti- CD3 (26). Similarly, anti-CD28 augmented the proliferation of CD28, and IL-4 augmented the proliferation induced by these PBMC from IL-2-treated patients induced by anti-CD3. In mitogenic antibodies. addition, the combination of anti-CD3, anti-CD28, and IL-4 The mechanism by which IL-2 therapy alters the proliferative further enhanced proliferation. Thus, although anti-CD28 is response of PBMC to 11-4 is not known. IL-4 induces the not a primary activation signal, it may be useful for further proliferation of lymphocytes activated by antigen or mitogen enhancing . (15-17) or by preincubation with IL-2 in vitro (22). Thus, PBMC represent a heterogeneous cell population, including systemic IL-2 and/or other intermediary cytokines produced both T-cell and non-T-cell populations. Because IL-4 is multi- endogenously in response to systemic IL-2 (30) are likely to potent, with effects on a variety of hematopoietic cell lineages result in preactivation of PBMC, including the observed in (9-17), its observed effects may be on both T- and non-T-cell crease in IL-2 receptor expression (5). IL-4 appears to down- populations. Similarly, anti-CD3, although a specific T-cell regulate the responsiveness of normal, resting lymphocytes to mitogen, may potentially induce proliferation of non-T-cells IL-2, possibly by inhibiting the increase in IL-2 receptor expres through the release of cytokines. Since accessory cells are sion (22). By contrast, IL-4 does not inhibit, but augments important in the proliferative response of lymphocytes to proliferation of PBMC preactivated by IL-2 which already have anti-CD3 (23), one potential mechanism by which IL-4 might increased expression of IL-2 receptors. IL-4 also enhances the augment the proliferative response to anti-CD3 might be by proliferation induced by mitogens which may directly enhance enhancing accessory cell function. However, IL-4 augmented the expression of IL-2 receptors. Thus, IL-4 may represent a anti-CD3 in the absence of accessory cells when immobilized regulatory factor which promotes the proliferation of activated anti-CD3 was used, indicating that the effects of IL-4 were cells, but inhibits IL-2-induced proliferation of bystander cells more directly on T-cells. Thus, the proliferative effects of IL-4 by inhibiting IL-2 receptor expression. and anti-CD3 appear to have been on the T-cell fraction of The proliferative response of PBMC from IL-2-treated pa PBMC preactivated by IL-2 in vivo, and the enhancement of tients to anti-CD3 appeared to be less than that of PBMC from proliferation by IL-4 was a direct effect of IL-4 on T-cells. healthy controls. This is unlikely to be due to a decrease in The proliferative effects of IL-4 alone and in combination circulating T-cells, since previous studies have shown that, after with anti-CD3 ±anti-CD28 suggest a potential use of these continuous-infusion IL-2 therapy, there is a rebound expansion agents for augmenting the generation of ex vivo activated cells of peripheral T-cells (5, 30) with no significant change in the for cancer therapy. IL-4 has recently been shown to induce percentage of CD3+, CD4+, or CD8+ T-cells (5). Since the LAK activity in PBMC preactivated by IL-2 (21, 22), and thus lymphocyte proliferation induced by anti-CD3 is mediated by it could potentially be used with or without anti-CD3 for LAK autocrine lymphokine production (23), the decreased prolifer cell generation in short- or long-term culture (25). IL-4 may 1163 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. IL-4 ENHANCEMENT OF PROLIFERATION AFTER II.-2 THERAPY

13. Nagler. A., Lanier. L. L.. and Phillips. J. H. The effects of IL-4 on human also be useful for the ex vivo expansion of other potentially natural killer cells: a potent regulator of IL-2 activation and proliferation. J. therapeutic cells (33). IL-4 augments the generation of specific Immunol.. 141: 2349-2351, 1988. cytotoxic T-cell responses (18-20), and IL-4 can support the 14. Grabstein. K. H., Park. L. S.. Morrissey. P. J.. Sassenfeld, H.. Price. V., growth and tumor-specific cytolytic activity of TIL cultured in Urdal. D. L.. and Widmer. M. B. Regulation of murine T-cell proliferation by B-cell stimulatory factor-1. J. Immunol.. 139: 1148-1153. 1987. IL-2 (34). The combination of IL-4 and anti-CD3 ±anti-CD28 15. Fernandez-Botran. R.. Sanders. V. M.. Oliver. K. G.. Chen. Y., Krammer. could potentially increase the cell number and/or function of P. H., Uhr, J. W., and Vitella. E. S. Interleukin-4 mediates autocrine growth of helper T-cells after antigenic stimulation. Proc. Nati. Acad. Sci. USA, 83: in vitro cultured TIL. Finally, since a limitation of systemic IL- 9689-9693. 1986. 2 therapy of cancer is dose-related toxicity (4, 5), the ability of 16. Spits. H., Yssel, H.. Takebe. Y.. Arai, N., Yokota, T.. Lee, F., Arai, K., IL-4 to augment the proliferation and LAK activity (21 ) induced Banchereau. J., and DeVries. J. E. Recombinant interleukin-4 promotes the growth of human T-cells. J. Immunol.. 139: 1142-1147. 1987. by IL-2 could potentially be exploited by using the systemic 17. Mitchell. L. C.. Davis. L. S.. and Lipsky. P. E. Promotion of human T- administration of IL-4 in combination with lower, less toxic lymphocyte proliferation by IL-4. J. Immunol.. 142: 1548-1557. 1989. doses of IL-2. Such studies must await the results of ongoing 18. Widmer, M. B., Acres, R. B., Sassenfeld, H. M., and Grabstein, K. H. 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1164 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. Enhancement by Interleukin 4 of Interleukin 2- or Antibody-induced Proliferation of Lymphocytes from Interleukin 2-treated Cancer Patients

Jonathan Treisman, Carl M. Higuchi, John A. Thompson, et al.

Cancer Res 1990;50:1160-1164.

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