Protective role of IL-2 during activation of T cells with bryostatin 1

International Journal of Immunopharmacology 22 (2000) 645±652 www.elsevier.com/locate/ijimmpharm

Protective role of IL-2 during activation of T cells with bryostatin 1 Ferdynand J. Kos a,c,*, David L. Cornell b, Anne B. Lipke b, Laura J. Graham b, Harry D. Bear b,c a

Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298, USA b Department of Surgery, Virginia Commonwealth University, Richmond, VA 23298, USA c Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA Received 24 September 1999; accepted 29 February 2000

Abstract Pharmacologic agents such as bryostatin 1 (bryostatin) can regulate cell activation, growth, and di€erentiation by modulating the activities of protein kinase C isoenzymes. Inhibition of growth of tumor cells and activation of T lymphocytes in vitro are the most recognized consequences of bryostatin treatment. The e€ect of bryostatin on T cells ranges from induction of apoptotic cell death to T cell activation, expansion, and acquisition of antigen-speci®c e€ector functions. Here, we describe the conditions under which these wide ranging e€ects occur. Mouse mammary tumor 4TO7-IL-2-primed lymph node cells exposed ex vivo to bryostatin upregulated CD25 expression but lost the ability to secrete IL-2. Most of these cells died by apoptosis unless IL-2 was provided for the duration of bryostatin treatment. Analysis of T cell repertoire by screening of T cells for the expression of di€erent Vb T cell receptor (TCR) families revealed that bryostatin-induced T cell death was unbiased and Vb-nonspeci®c. Within particular Vb clones, only CD25+ T cells survived exposure to bryostatin and IL-2. Treatment of 4TO7 tumor-bearing mice with a single injection of low dose bryostatin followed by multiple low doses of IL-2, but not with bryostatin alone, delayed tumor growth. These results indicate that activation of T cells with bryostatin should be carried out under protection of exogenous IL-2 to ensure survival and expansion of T cells that may exhibit anti-tumor activity. 7 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Bryostatin; CD25; IL-2; T cell activation; Tumor immunotherapy

1. Introduction * Corresponding author. Tel.: +1-804-828-8779; fax: +1804-828-8453. E-mail address: [email protected] (F.J. Kos).

Bryostatins constitute a family of agents displaying unique biological features ranging from direct antitumor e€ects [1±3] to immunomodula-

0192-0561/00/$20.00 7 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 2 - 0 5 6 1 ( 0 0 ) 0 0 0 2 7 - 8

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tory activities [4±9]. It is thought that bryostatins primarily modulate the enzyme activity of serine/ threonine kinase protein kinase C (PKC), a critical regulator of cell activation and growth. Bryostatin 1 (bryostatin) is a natural macrocyclic lactone isolated from the marine invertebrate Bugula neritina [1]. Bryostatin binds to, activates, and downregulates some PKC isoenzymes by causing their degradation [10±15]. This downregulation of PKC may lead to inhibition of downstream transcription factors and matrix metalloproteinases which are involved in cancer development. In addition, treatment of tumor cells with bryostatin may lead to their death by apoptosis [16]. All these activities of bryostatin have renewed interest in its potential for cancer therapy. Bryostatin has already undergone phase I and II clinical trials in humans [17± 21] and further testing is being continued. The e€ect of bryostatin on T lymphocytes is still poorly understood. This issue, however, deserves further attention if we are to exploit the immunomodulatory properties of this agent or to protect against immunotoxicity caused by bryostatin in vivo. For the past several years we have using bryostatin in combination with IL-2 for activation and expansion of T cells for tumor adoptive immunotherapy [6,22]. More recently, we have modi®ed our approach towards adoptive immunotherapy with T cells by using a strategy based on short-term ex vivo treatment (``pulsing'') of tumor-sensitized T cells with bryostatin and IL-2 before adoptively transferring them to tumor-bearing hosts that are subsequently treated with low doses of IL-2. This procedure eliminates a prolonged and cumbersome step of expanding T cells in culture before adoptive transfer, however, it does not compromise the immunotherapeutic potential of these T cells (manuscript in preparation) [8]. Since the requirement of IL-2 during activation of T cells with bryostatin was surprising, we have analyzed the role of IL-2 in this process. 2. Materials and methods 2.1. Mice Female BALB/c (H-2d) mice were purchased

from Charles River Laboratories (Wilmington, MA). Six- to 16-week old mice were used in these experiments. 2.2. Cell lines Mouse mammary carcinoma cell lines 4TO7 (H-2d) and 4TO7-IL-2, i.e., 4TO7 transfected with IL-2 gene [23] (both lines obtained from Dr. Fred Miller, Breast Cancer Program, Karmanos Cancer Institute, Detroit, MI) were cultured in Dulbecco's modi®ed Eagle medium (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies, Grand Island, NY), 10% NCTC 109 medium (Sigma Chemical, St. Louis, MO), 8 mg/ml bovine crystalline insulin (Sigma), 1 mM oxaloacetic acid, nonessential amino acids, and antibiotics (100 U/ml penicillin and 100 mg/ ml streptomycin) at 378C in a humidi®ed atmosphere of 5% CO2 in air. 2.3. Preparation of tumor-sensitized lymph node cells and their ex vivo treatment with bryostatin and IL-2 BALB/c mice were injected into their hind footpad with 1  106 4TO7-IL-2 tumor cells. Ten days later, draining popliteal lymph nodes were removed and single cell suspensions were prepared. Lymph node cells at a ®nal concentration of 1  106 per ml were incubated for 16±20 h in culture medium (RPMI 1640, Life Technologies; 10% FBS; antibiotics) in 50 ml conical polypropylene tubes in the presence of 5 nM (5 ng/ml/1  106 cells) bryostatin 1 (manufactured by Ben Venue Laboratories, Bedford, OH; provided to us by the National Cancer Institute, Bethesda, MD), IL-2 (Chiron, Emeryville, CA) at 40 U/ml, or their combination. 2.4. Analysis of CD25 and TCR Vb expression on bryostatin-treated lymph node cells by ¯ow cytometry Tumor-draining lymph node cells, prepared as above, were washed, stained with monoclonal antibodies (mAbs), and analyzed by ¯ow cytome-

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try using an EPICS XL-MCL (Coulter, Miami, FL) instrument. The following mAbs, purchased from PharMingen (San Diego, CA), were used: PE-conjugated rat anti-mouse CD4, CD8, and CD25 (IL-2 receptor (IL-2R) a chain), FITCconjugated rat anti-mouse Vb2 TCR, Vb3 TCR, Vb6 TCR, and FITC-conjugated mouse antimouse Vb8.1, 8.2 TCR and Vb12 TCR. In some experiments, T cells were enriched from pooled lymph node cell preparations by nonadherence to nylon, as described [24]. For TCR Vb analysis, enriched T cell populations were treated with bryostatin, IL-2, and bryostatin/IL2 for 16±20 h, as described in the Section 2.3. After washing, the cells were left in culture for another 20 h before staining for ¯ow cytometry with anti-CD4 plus anti-CD8 and anti-Vb TCR. 2.5. Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay To detect and quantitate apoptotic cell death in tumor-draining lymph node cells treated with bryostatin, IL-2, or their combination, the TUNEL technique followed by ¯ow cytometric analysis was used. Brie¯y, ex vivo treated lymph node cells, prepared as described above, were ®xed with 2% paraformaldehyde solution in phosphate bu€ered saline in V-bottomed 96-well plates, permeabilized with a solution of 0.1% Triton X-100 in 0.1% sodium citrate, and labeled with a ¯uorescein TUNEL reaction mixture using the in situ cell death detection kit (Boehringer Mannheim, Mannheim, Germany). The assay was performed according to the protocol provided by the manufacturer. Cells used as negative controls were ®xed, permeabilized, and treated with a label solution containing ¯uoresceinlabeled nucleotides without terminal transferase. Positive controls were samples of cells, ®xed, permeabilized, and treated with DNase I (Sigma) for 10 min at room temperature to induce DNA strand breaks followed by TUNEL reaction mixture with enzyme solution and labeled nucleotides. Flow cytometric analysis was performed on an EPICS XL-MCL (Coulter) instrument.

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2.6. Analysis of IL-2 production by bryostatintreated lymph node cells Tumor-draining lymph node cells were prepared and pulsed as above. After washing, the cells were set up in 24-well plates (2  106/ml/ well) and incubated for 48 h. Collected culture supernatant was stored at ÿ708C until use. IL-2 concentration in culture supernatants was quantitated using mouse IL-2 ELISA kit (Endogen, Cambridge, MA). 2.7. Treatment of 4TO7-bearing mice with bryostatin and IL-2 Solid ¯ank tumors were established in BALB/c mice by i.d. injections of 2.5  105 4TO7 cells. Treatment was initiated on day 3 via i.p. injections of a single 1 ng dose of bryostatin followed by human recombinant IL-2 (Chiron) given at a dose of 5000 IU i.p. twice a day for 5 days. Bidirectional tumor measurements were recorded twice a week and expressed as tumor area in square mm.

Table 1 Expression of CD25 on 4TO7-IL-2-primed lymph node cells untreated (NIL) and treated ex vivo for 20 h with bryostatin (B), IL-2, and bryostatin in combination with IL-2 (B/IL-2)a Treatment

NIL B IL-2 B/IL-2 a

% CD25+ cells from 4TO7-IL-2-primed lymph nodes Lymph node cells

T cells

Exp. 1

Exp. 2

Exp. 1

Exp. 2

17.1 37.2 18.2 52.4

9.4 19.5 12.0 41.0

24.7 51.4 21.8 77.2

16.0 32.1 15.6 49.2

Tumor-sensitized popliteal lymph node cells were prepared and treated ex vivo as described in Section 2.3. Cells were stained for ¯ow cytometry as described in Section 2.4. Percentage of CD25+ cells within either unseparated lymph node cells pooled from ten mice or T cells enriched to 93 2 4% purity from lymph node cell populations by nonadherence to nylon (24), is given. The results of two independent experiments (Exp. 1 and Exp. 2) are shown.

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3. Results To study the role of IL-2 in bryostatin-induced T cell activation, we performed a series of in vitro experiments. Initially, 4TO7-IL-2-primed lymph node cells stimulated ex vivo with bryostatin, IL-2, or their combination were analyzed by ¯ow cytometry. 4TO7-IL-2-primed lymph node cells (10 days after immunization) contain a signi®cant proportion of activated cells, including T cells. We observed that 52±70% of TCRb+ T cells were CD44+ cells, and 15±25% of T cells were CD25+ (IL-2R+). Interestingly, a 20 h treatment of lymph node cells with bryostatin alone doubled the percentage of CD25+ cells, and a combination of bryostatin and IL-2 resulted in tripled percentage of CD25+ cells. The same proportional increase was noted for puri®ed T cells (Table 1). Since bryostatin is a known promoter of apoptosis, we analyzed 4TO7-IL-2-primed and ex vivo stimulated lymph node cells to detect apoptotic cell death. Representative results, shown in Fig. 1, reveal that lymph node cells (or puri®ed T cells) stimulated with bryostatin alone undergo apoptosis. Primed lymph node cells treated for 16 h with bryostatin alone die by apoptosis (84% of

cells show DNA degradation) unless rescued by the addition of IL-2 (9% of cells show DNA degradation) (Fig. 1). This observation along with the data on upregulation of IL-2R suggest that bryostatin might compromise IL-2 production by T cells. Indeed, the analysis of IL-2 production ex vivo by 4TO7-IL-2-primed lymph node cells has shown that their exposure to bryostatin alone inhibits IL-2 secretion (Fig. 2). When tumor-primed lymph node cells are treated with bryostatin in combination with IL-2, these cells still do not produce IL-2. These results correlate well with those presented in Fig. 1 and Table 1; the presence of IL-2 during the contact of IL-2R-expressing T cells with bryostatin rescues them from apoptotic cell death and promotes their further expansion. Our preliminary analysis has shown that 4TO7-IL-2 induces expansion of tumor-draining lymph node T cells expressing TCRs with Vb8.1/ 8.2, Vb2, Vb3, Vb6, and Vb12, but not with the following Vbs: 4, 5, 7, 9, 10, 11, 13, and 14 (data not shown), as compared to naiÈve lymph node T cells. To test whether exposure of tumor-draining lymph node T cells to bryostatin a€ects Vb repertoire selectively, we phenotyped TCR Vbs after treatment of enriched T cell populations

Fig. 1. Analysis of apoptotic cell death in 4TO7-IL-2-primed lymph node cells untreated (NIL) or treated for 16 h with bryostatin (B), IL-2, or B/IL-2. The TUNEL assay and analysis by ¯ow cytometry were performed as described in Section 2. Percentage of positive cells, given in the corner of each graph, was calculated against a negative control incubated with label solution in the absence of terminal transferase. Data are representative of two independent experiments.

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Fig. 2. Analysis of IL-2 production by 4TO7-IL-2-primed lymph node cells untreated (NIL) and treated for 16 h with bryostatin (B), IL-2, or B/IL-2. IL-2 concentration in supernatants from 48 h cultures of lymph node cells was determined by ELISA. Results are mean values 2 SE of triplicate cultures.

with bryostatin, IL-2, or bryostatin/IL-2. Representative results from these experiments, shown in Fig. 3A, indicate that bryostatin-mediated e€ects are not Vb-speci®c. In general, no substantial decrease or increase in the percentage of Vb+ cells was observed as a result of exposure of T cells to bryostatin alone, except for Vb3+, Vb12+, and Vb8.1/8.2+ cells. A number of Vb3+ cells remained at the level of untreated controls if IL-2 accompanied bryostatin during T cell treatment, suggesting that IL-2 rescued these cells from apoptotic death. In contrast, IL-2 combined with bryostatin was unable to rescue Vb12+ cells from their decrease in number, suggesting that these cells did not respond to IL2 (Fig. 3A). Correlation of these results with the data showing the expression of CD25 (Fig. 3B) o€ers an explanation for di€erent responses of Vb12+ and Vb3+ cells to bryostatin/IL-2 treatment. In contrast to most Vb3+ cells, Vb12+ cells did not express CD25 and did not respond to IL-2. Thus, it seems that T cells that expressed CD25 before treatment (Vb3+ and Vb8.1/8.2+ cells) and T cells that upregulated CD25 expression during B/IL-2 treatment (Vb2+ and Vb6+ cells) respond to IL-2 and are protected from bryostatin-induced apoptosis (Fig. 3). An additional issue that should be addressed in these studies is how bryostatin versus bryostatin/IL-2 treatment a€ects antigen-speci®c T cells. Since no tumor-speci®c antigens on 4TO7 cells are known yet, this experimental system is not suitable to

Fig. 3. E€ect of bryostatin on the Vb repertoire of T cells from tumor-draining lymph nodes. Enriched T cell populations from 4TO7-IL-2-primed lymph nodes were treated ex vivo for 16 h with bryostatin (B), IL-2, bryostatin and IL-2 (B/IL-2), or left untreated (NIL). After washing, the cells were cultured for another 20 h and stained for ¯ow cytometry with anti-CD4 plus anti-CD8 and with mAbs which recognize indicated Vb TCRs: (A) shows percentage of T cells (CD4+ plus CD8+) expressing a particular dominating TCR Vb element. (B) shows percentage of CD25+ cells (mean 2SE) within indicated untreated (NIL) or B/IL-2-treated Vb+ primed T cells. Results are representative of two di€erent experiments.

analyze the e€ect of bryostatin on antigen and Vb speci®cities. We asked the question whether bryostatin alone, or in combination with IL-2 directly injected into tumor-bearing mice exerts a therapeutic e€ect. Fig. 4 shows that a single low dose (1 ng/mouse) of bryostatin alone does not a€ect 4TO7 tumor growth. A high dose of bryostatin (1 mg/mouse) signi®cantly accelerated tumor growth (data not shown). Interestingly, 1 ng single dose of bryostatin in combination with low dose of IL-2 given twice a day for 5 days, but not IL-2 alone, signi®cantly inhibited 4TO7 tumor growth, although this never caused complete tumor regression (Fig. 4). These results suggest a requirement for IL-2 in maintaining

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bryostatin-induced e€ects. Clearly, direct administration of bryostatin and IL-2 to tumor-bearing mice does not have the same powerful e€ect as does the transfer of tumor-primed and ex vivo activated lymph node cells (data not shown). It is possible that this treatment protocol is not yet completely optimized or that associated toxicity of systemic treatment reduces the immunotherapeutic e€ect of activated T cells. 4. Discussion Mouse mammary tumor 4T07 is a weakly immunogenic tumor whose growth is strongly inhibited in primed mice [25]. IL-2-secreting 4TO7-IL-2 tumor induces active immunity in mice that leads to its spontaneous rejection [23]. Speci®c anti-4TO7 immunity is in part mediated by CD8+ T cells and their depletion in vivo abrogates speci®c immunity against 4TO7 cells and accelerates tumor growth (unpublished observation). When 4TO7-IL-2 primed lymph node cells are transferred to 4TO7 tumor-bearing mice, they do not induce complete tumor regression unless pretreated with bryostatin in the presence of IL-2 (manuscript in preparation). The results of this study suggest that activated T cells (from tumor-draining lymph nodes) treated with bryostatin lose the ability to secrete IL-2 and most of them die by apoptosis unless provided with exogenous IL-2. This observation corroborates the results of studies showing that bryostatin alone is not mitogenic to T cells and, to the contrary, prolonged exposure of T cells to bryostatin is toxic [5,12,26]. The mechanism responsible for this e€ect is not known, but it may be related to the fact that downregulation of some PKC isoenzymes inhibits IL-2 synthesis. Recently, Szamel et al. reported that neutralization of PKC-b, -d, and -e inhibited IL-2 synthesis, whereas blockade of PKC-a and -y inhibited IL-2R expression [27]. In our recent study we have already presented evidence that PKC-d is one of the primary targets of bryostatin, which selectively downregulates this isoenzyme [15]. Thus, it is possible that degradation of PKC-d by bryostatin contributes to the inhibition

Fig. 4. E€ect of bryostatin alone versus bryostatin/IL-2 treatment on 4TO7 tumor growth in vivo. Four groups of mice were inoculated with 4TO7 tumor cells. Treatment was initiated on day 3 with a single 1 ng dose of bryostatin followed by IL-2 at 5000 units twice a day for 5 days (open squares, B/ IL-2). Control mice were untreated (closed squares, NIL) or treated with bryostatin alone (open triangles, B) or IL-2 alone (open circles, IL-2). Mean values of tumor size from 5 animals 2SE are shown as a function of time. Similar results were obtained in three separate experiments.

of IL-2 production by activated T cells which subsequently, in the absence of their primary growth factor, die by apoptosis. At the same time, delivery of exogenous IL-2 to activated T cells with upregulated IL-2Rs overcomes the defect caused by bryostatin and ensures their survival and further expansion. Murine activated and resting memory CD8+ T cells express IL-2R but at very low levels [28,29]. It has also been shown that a proportion of putatively IL-2Rÿ memory CD8+ cytotoxic T cell precursors requires IL-2 for their development into e€ectors [30]. Although it is possible that other cytokines, e.g., IL-7 can contribute to the activation of resting memory T cells (including IL-2Rÿ) in an IL-2-independent manner [28,31], an interesting possibility is that inducible and rapid IL-2R upregulation precedes recall T cell responses. Undoubtedly, a simple pharmacological approach (bryostatin?) that would upregulate IL-2R and predispose resting memory T cells to

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respond to restimulation with antigen may o€er a desirable step in the induction of anti-tumor immune responses. TCR-initiated antigen-speci®c signaling for T cell activation starts with an increase in hydrolysis of inositol phospholipids and production of diacylglycerol and inositolphosphates, which in turn can increase intracellular calcium concentration and activate PKC isoenzymes. PKCs phosphorylate and regulate various downstream targets, including several transcription factors, and in this way regulate T cell activation genes [32,33]. Pharmacologic agents such as phorbol esters or bryostatin can partially mimic the e€ects of diacylglycerol and a€ect PKC-regulated T cell functions. Similarly to NK cells [15], distinct T cell functions can be regulated by di€erent PKC isoenzymes [27,34]. Attempts have already been made to compare the expression pro®les of PKC isoenzymes in naiÈve versus activated/memory T cells [35±37]. Since the di€erences in qualitative and quantitative expression of PKC isoenzymes were observed, we can speculate that functional di€erences between those two cell types arise from the di€erences in signaling properties. If so, these subsets of cells can be differentially regulated with pharmacologic agents that selectively target responsive PKC isoenzymes, such as bryostatin or phorbol esters. The idea that antigen-speci®c secondary T cell responses can be activated with a pharmacologic agent and without the antigen is not new and should no longer be considered unrealistic. More than ten years ago, a study from Doherty's laboratory documented that lymphocytic choriomeningitis virus-speci®c CD44+ memory T cells can be selectively activated to proliferate and mediate cytotoxic activity following treatment with PMA, ionomycin, and IL-2 [38]. Our studies take this idea one step further, showing that tumor-sensitized T cells after treatment with PKC modulator, bryostatin, acquire anti-tumor activity if the appropriate conditions allowing their activation and survival are ensured.

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Acknowledgements This work was supported by the National Institutes of Health grant CA48075 (to H.D.B) and by the Cancer Immunology Research Foundation of Concern Foundation grant (to F.J.K.).

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