Phenotype Analyses and Cellular Mechanisms of the Pre-effector T-Lymphocyte Response to a Progressive Syngeneic Murine Sarcoma1

Lymph nodes draining the progressively growing, weakly immunogenic, MCA 105 sarcoma contained tumor-sensitized but not fully func tional pre-effector T-cells. These cells could further differentiate to acquire full antitumor effector function for adoptive therapy in an estab lished in vitro sensitizaron (IVS) procedure. In this study, we utilized selective depletion with antibodies of lymphocyte subsets bearing the L3T4 «'1)4) or Lyt-2 (CD8) antigen and of cells bearing the asialo-GM, (ASGM-1) glycosphingolipid to identify the phenotype of pre-effector cells elicited during progressive tumor growth. Cells from lymph nodes draining a progressive MCA 105 tumor in the footpad were treated with antibodies plus complement prior to IVS. The antitumor efficacy of resulting IVS cells was assessed in adoptive therapy of 3-day established pulmonary MCA 105 métastases. Depletion of 1.yt-2"*cells eliminated in vivo antitumor reactivity with concurrent elimination of in vitro cytotoxic activity against the MCA 105 tumor, whereas depletion of 1314* cells did not have an impact on either in vivo or in vitro antitumor reactivities. Treatment with ASGM-1 antiserum plus complement was also found to abrogate therapeutic efficacy. However, the in vitro cytotoxic activity was not affected. These results indicate that the pre-effector cells were Lyt-2+, L3T4", and ASGM-1*. We next examined whether the sensitization of pre-effector cells in vivo required the participation of L3T4* helper cells. To approach this, mice were depleted of L3T4*, Lyt-2+, or ASGM-1+ cells by antibody injections before tumor inoculation. Treat ment with Lyt-2 monoclonal antibody abrogated the pre-effector cell response in the draining lymph nodes, as evidenced by failure to generate therapeutically effective cells following IVS. On the other hand, neither L3T4 nor ASGM-1 antibody treatment affected the generation of preeffector cells. Thus, sensitization of Lyt-2+ pre-effector cells in response to progressive tumor occurred in the absence of L3T4* helper cells. INTRODUCTION The most critical component of successful adoptive immunotherapy of cancer is the identification and isolation of lym phocytes with potent antitumor effects. In studies with transplantable tumors, lymphocytes from hyperimmunized syngeneic animals represent such cell populations and provided a convenient source of antitumor effector cells (1-5). However, this immunization technique of obtaining immunologically re active lymphocytes, although helpful in demonstrating the po tential of cellular therapy, is poorly suited for use in humans. It has, therefore, been predicted that the application of adoptive immunotherapy for human cancer will require the development of culture techniques that can allow stimulation and expansion of antitumor lymphocytes from cancer patients. The discovery of T-cell growth factor, now termed IL-2,4 and Received 10/24/89; revised 2/2/90. 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 in part by Grant 1M-494 from the American Cancer Society and USPHS Grant CA-49231 from the National Cancer Institute, NIH. 1Present address: First Department of Surgery, University of Tokyo, Tokyo, Japan. 3To whom requests for reprints should be addressed, at 1520 MSRB I, Box 0666, Department of Surgery, University of Michigan, Ann Arbor, MI 481090666. 4The abbreviations used are: IL-2, interleukin 2; LN, lymph nodes: HBSS, Hanks' balanced salt solution; IVS, in vitro sensitization (sensitized); ASGM-1, asialo-GM,; CM, complete medium; Rig, rat immunoglobulins; mAb, monoclo nal antibodies; NRS, normal rabbit serum; NK, natural killer; LAK, lymphokineactivated killer; FMF, flow microfluorimetry; B6, C57BL/6J. subsequent availability of large quantities of purified recombi nant material seemed to have provided a means for in vitro expansion of sensitized T-cells (6, 7). Early experiments using the highly immunogenic FBL-3 leukemia have demonstrated the therapeutic efficacy of IL-2-expanded immune lymphocytes (8, 9). More recently, we have established another culture system utilizing IL-2 for propagating immune T-lymphocytes with reactivity against the weakly immunogenic MCA 105 and MCA 106 sarcomas (10). The development of this IVS tech nique has helped to establish conditions necessary for expand ing T-lymphocytes while maintaining their antitumor reactivity. This method was originally thought to facilitate clonal expan sion of previously sensitized immune effector cells. However, phenotype analyses of cells before and after IVS revealed that, in addition to cellular expansion, the culture system induced differentiation of antitumor effector cells (11). Because of these observations, an attempt was made to ex plore the feasibility of generating therapeutically effective lym phocytes from mice bearing progressive tumors. Unlike lymphoid cells from tumor-immunized animals, freshly isolated lym phocytes from tumor-bearing mice exhibited little or no antitumor effects when tested in adoptive immunotherapy ex periments. However, after secondary IVS, recovered cells dem onstrated potent in vivo antitumor reactivity (12-14). Since similar cells could not be generated from normal lymphocytes, progressive tumor growth must have triggered the production of cells that were sensitized to the tumor but not fully functional immunologically. These cells were then referred to as "preeffector" cells. In a previous study, the antitumor effector cells generated by IVS have been identified as belonging to the Lyt-2 phenotype of T-lymphocytes (12). However, the nature of pre-effector cells elicited in response to tumor growth was poorly defined. In this study, we have used the MCA 105 sarcoma to analyze the phenotype of pre-effector cells and the cellular requirement during IVS for induction of functional effector lymphocytes. More importantly, we utilized a previously established method of in vivo depletion of lymphocyte subsets to investigate the immunological mechanisms underlying the sensitization of preeffector cells during progressive tumor growth. MATERIALS AND METHODS Mice. Female B6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Caged in groups of 6 or fewer, they were maintained in a specific pathogen-free environment and were used for experiments at age 8 to 12 weeks. Tumor. The MCA 105 sarcoma is a 3-methylcholanthrene-induced tumor of B6 origin (5). A large number of vials of this tumor from the first in vivo passage were cryopreserved. After thawing, the tumor was maintained in syngeneic mice by serial s.c. transplantation. The MCA 105 sarcoma used for this study was in the fourth to seventh transplan tation generation. Another similarly induced syngeneic tumor, MCA 102 sarcoma, was used as control tumor in the cytotoxicity assay. Single-cell suspensions were prepared from solid tumors by digestion with a mixture of DNase, collagenase, and hyaluronidase, as previously described (12). For establishing solid tumor growth, B6 mice were 4371 on April 14, 2017. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from IMMUNE RESPONSE TO PROGRESSIVE TUMOR inoculated s.c. in the footpad with 2 to 4 x IO5 MCA 105 tumor cells in 0.05 ml of HBSS (GIBCO, Grand Island, NY). The growth of tumors became evident on day 3 and all animals eventually succumbed to the progressive tumor, with a median survival time of 23 days. LN Cell Preparation. Popliteal LN draining the progressive tumor were removed from mice 12 to 15 days after tumor inoculation, when the footpad tumor reached a thickness of 6 to 8 mm. Single-cell suspensions were prepared mechanically by pressing LN with the blunt end of a 10-ml plastic syringe in HBSS. The resulting cell suspensions were filtered through a layer of no. 100 nylon mesh (Nitex; Lawshe Industrial Co., Bethesda, MD), washed, and resuspended for IVS. IL-2. The recombinant human IL-2 was kindly supplied by the Cetus Corporation (Emeryville, CA). The biological and immunological prop erties of IL-2 have been described in detail (7). Purified material had a specific activity of 4 to 8 x IO6 units/mg protein (Cetus units). The endotoxin content was <0.1 ng/106 units IL-2, as assessed by the standard Limulus assay. IVS Procedure. The procedure for IVS of tumor-draining LN cells has been previously described (13). In brief, 2 x IO5 responding LN cells and 4 x 10s irradiated (2000 rad) MCA 105 stimulator tumor cells were cultured in 2.0 ml CM containing 10 units/ml IL-2 in wells of 24-well tissue culture plates (Costar, Cambridge, MA). The compo sition of CM has been described elsewhere (15). The cultures were incubated at 37°Cin 5% CO2 and were fed with 1.0 ml of CM with 10 units/ml IL-2 on day 5. The IVS cells were routinely harvested on days 8-9, when they grew to a high density. These cells were washed 3 times before resuspending in HBSS for adoptive immunotherapy or in CM for cytotoxicity assay. Adoptive Immunotherapy. In vivo antitumor effects of IVS cells were assessed by adoptive therapy of established pulmonary MCA 105 métastases. B6 mice were given i.v. injections of 2 to 3 x 10' MCA 105 tumor cells in 1.0 ml HBSS, to establish pulmonary métastases. In all adoptive immunotherapy experiments, recipient mice were immunosuppressed by i.v. injections of 100 mg/kg cyclophosphamide (Sigma Chemical Co., St. Louis, MO) 6 h prior to tumor inoculation (16). Although not necessary, this pretreatment facilitated the establishment of consistent numbers of pulmonary tumor nodules between individual animals. In addition, our previous findings clearly demonstrated that host immunosuppression had no impact on the ability of transferred cells to mediate the regression of pulmonary MCA 105 métastases (17). On day 3, IVS cells were given i.v. and, from days 3 through 5, the mice received i.p. injections of 7500 units IL-2 every 12 h. This regimen of IL-2 alone had little or no antitumor effect but could enhance the therapeutic efficacy of transferred IVS cells (10-12). On days 12 to 14 after tumor inoculation, the mice were sacrificed for enumeration of pulmonary nodules by counter-staining of the lung with 15% india ink solution, as described (18). Lungs with métastasestoo numerous to count on autopsy were assigned an arbitrary value of 250, because this was the largest number of tumor nodules that could be reliably counted in each lung. Cytotoxicity Assay. The 4-h 5'Cr-release assay was carried out as previously described (13). Briefly, 1 x 10" "Cr (Na25'CrO4; New Eng land Nuclear, Boston, MA)-labeled target cells and various numbers of effector cells were incubated together in 0.2 ml CM in a round-bottomed microtiter well at 37°Cfor 4 h. Cell-free supernatant was harvested with the Titertech Collecting System (Flow Laboratories, Inc., McLean, VA). The percentage of 5'Cr release was calculated by using the follow ing formula: (experimental release spontaneous release)/(maximal release —spontaneous release) x 100. Depletion of Lymphocyte Subsets in Vitro and in Vivo. Two hybridomas producing rat IgG2b mAb against the mouse L3T4 (GK1.5) and Lyt-2 (2.43) T-cell antigens were obtained from American Type Culture Collection (Rockville, MD). The mAb were produced as ascites fluid from sublethally irradiated (500 rad) DBA/2 mice. The rabbit antiserum to ASGM-1 was purchased from Wako Chemicals (Dallas, TX). For in vitro depletion of L3T4+ or Lyt-2+ cells, LN cells were suspended at a concentration of 107/ml in cytotoxicity medium containing a 1/100 dilution of mAb ascites fluid. Cytotoxicity medium was composed of RPMI 1640 with 0.3% bovine serum albumin (Pathocyte 4; Miles Laboratories, Elkhart, IN) and 25 HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer. Cells were incubated at 4°Cfor 45 min, followed by incubation with an excess of a mouse anti-rat K-chain mAb (MAR 18.5; American Type Culture Collection) at 4°Cfor 45 min. They were then centrifuged and resuspended in 50% of the original volume with complement (Low-tox-M rabbit complement; Accurate Chemical and Scientific Corporation, Westbury, NY) at 1/8 dilution for 60 min at 37°C.Treatment with complement was repeated once and cells were washed before IVS culture. The procedure for in vitro depletion of ASGM-1+ cells from tumor-draining LN cells was essen tially the same as that for L3T4+ or Lyt-2+ cell depletions, except that the rabbit antiserum was used at 1/50 dilution and there was no additional treatment with the anti-rat mAb. For in vivo depletion, B6 mice received one i.v. injection of 0.2 ml of monoclonal ascites fluid or rabbit heteroantiserum diluted to 1.0 ml with HBSS, as described previously (19, 20). This antibody treatment of mice was done on the day of initiating intrafootpad MCA 105 tumor growth. FMF Analysis. Analysis of cell surface phenotypes was carried out by indirect immunofluoresence staining, analyzed by a FACScan flow microfluorometer (Becton Dickinson, Sunnyvale, CA). Single-cell sus pensions (5 to IO x 10" cells) were incubated for 45 min at 4°Cwith 20 ¡Aof appropriately diluted mAb in phenol red-free HBSS containing 2% fetal bovine serum and 0.1% NaN3. Bound antibodies were detected by incubation with 25 M'of the fluorescein isothiocyanate-labeled mAb to rat /(-chain (Phar Mingen, San Diego, CA) or fluorescein isothio cyanate-labeled goat anti-rabbit IgG (Pel-Freez, Rogers, AK). Fluores cence profiles were displayed as logarithmically increasing fluorescence intensity versus cell numbers. Statistics. The Wilcoxon rank-sum test (21) was used to determine the significance in differences in numbers of pulmonary tumor nodules between groups. Two-sided P values are presented.