Specific studies on in vitro generated cytotoxic T lymphocytes directed against human target cells grown in monolayer culture.

Specific cytotoxic T lymphocytes (CTL) against HLA antigens of skin fibroblasts were generated with mixed leukocyte culture (MLC). Nonspecific cytotoxicity (CTX) in 3H-proline microcytotoxicity test was detected as early as the 4th day by primary in vitro sensitization (IVS), and the activity continued until around the 8th day. The specific CTX by primary IVS was detectable from the 7th day to 12th day, and by secondary IVS, from around the 2nd to 4th day. However, obtainment of specific CTX only was rarely the case, and the probability of obtaining them did not increase until the 9th to 10th day by primary IVS and until the 3rd to 4th day by by secondary IVS. This is related to reduction of blastogenic response in MLC and suggests that inactivation of blast cells generated by MLC is of importance in obtaining specific CTX. A CTX assay was conducted using cryopreserved effector cells, which revealed that in most cases nonspecific CTX either decreases or disappears altogether. On the other hand, specific CTX activity (more than 30 % reduction of only test target cells) remained and the specific CTX pattern was good and evident in many cases. Specific CTL was generated against fibroblast antigens by adding allogenic lymphocytes as third party stimulators. Cryopreservation technique was effective in increasing the specificity of CTX. 49 INTRODUCTION destroyed in CMC assay, in which the target cells used were grown in suspension culture21 • '. MLC and cell-mediated cytotoxicity (CMC) studies have shown that lymphocytes cultured with allogeneic lymphocytes respond to allogeneic HLA antigens (HLA-D) by undergoing blast transformation, and subsequently CTL can be differentiated and directed against the target cell antigens present on the stimulating cells1•6l. Whereas target cells autologous to or scngenic with responding lymphocytes are not usually In practice, however, most human solid tumors are grown as monolayer culture cells in 1Jitro, and when the CMC tests are performed by using effector cells generated in vitro against monolayer tissue culture target cells, nonspecific CTX often develops. These monolayer culture target cells appear to be very sensitive to nonspecific cytotoxic effects of blast cells3' 5, 9l. Therefore, the present study was undertaken *' '.f:kll!'.ll.fU, lll*?J:l:f!.Et~. 1J':!E;;*IJfFf, ~rntft:tzT-, Micheal A. Bean, rfil*=f:!1j: l:'.. 1'~Jltrfr31ti%!H}fcgvr..x;J--tQi%!Hjfcgn ~tl:.T !J h~;E;j(O) in vitro ~lg Correspondence: Mitoshi Akiyama, M. D., Department of Pathology Radiation Effects Research Foundation 5-2 Hijiyama Park Minami-ku, Hiroshima 730, Japan 50 M. Akiyama et al. to establish selectively specific CTL generation by MLC against surface antigens expressed by human skin fibroblast target cells, and also to study methods of cryopreservation of effector CE>lls in order to check whether their function can be maintained. Cryopreservation has proven to be an effective way for reducing nonspecific killing cells and retaining function of specific CTX of effector cells. For the purpose of generating specific CTL to autologous tumor cells, the conditions necessary to generate CTL against monolayer cultured tumor target cells were also studied by using allogeneic fibroblasts as stimulator cells, since they (:\.re known to bear serologically defined (SD) HLA surface alloantigens just as tumor cells bear SD antigens. MATERIALS AND METHODS Details of HLA lo'cus antigens of the study subjects from whom lymphocytes and skin :fibroblasts were obtained are shown in Table 1. Isolation of human lymphocytes from peripheral bllod. It was performed as described elsewwhere10J. Briefly, human peripheral blood mononuclear cells were isolated from normal volunteers, who were also donors of skin fibroblast ~ell lines, by defibrination and use of Ficoll-Hypaque solution (specific density 1. 077 ±0. 001) followed by three washes before suspension in medium for IVS. The medium consisted of Eagle's minimal essential medium (MEM) containing 10% heat-inactivated fresh normal human serum which is usually obtained from stimulator donors in IVS, and 100 IU of penicillin and 100 pg streptomycin/ml (1.96' P /S), 1.% nonessential aminoacid (NEAA) and 2 mM fresh L-glutamine (1% L-glutamine). These Table 1. HLA locus antigens on lymphocytes and skin fibroblasts of donors Normal HLA Locus Antigens Donors Sex A B Cw Dw M.B. M 2 7, 12 5 3 c.s. M 2, 29 12 2 Y.K. M 2, 9 5 1 J.E. F 2, 10 12, 27 1 T.P. F 11, W24 12, 40 J.M. M 1, w24 8, 15 2, 3 M.C. F 2, 29 12 1 mononuclear cells were used as responder and stimulator cells for IVS. In vitro sensitization. One-way MLC was used to sensitize responder normal lymphocytes under various conditions against alloantigens on irradiated stimulating lymphocytes or fibroblasts (irradiated with °Co to 2, 250 rad and 4, 500 rad, respectively). MLC was performed in tissue culture flasks at several ratios of responder and stimulator cells and keeping the conditions similar to those used for MLC in microtest plate (Falcon # 3040) in order to maintain comparable culture conditions between the systems. The responder cell density and amount per medium surface area of culture flasks were kept constant (i.e., 125x103 responding cells in 0. 2 ml of test medium per 0. 282 cm2 of surface area of culture flasks). Unless otherwise noted, half volume of culture medium in the flasks was usually replaced every three days with fresh test medium. Harvesting MLC cells from flasks and checking mixed leukocyte response by 3 H-thymidine uptake. Eighteen hours before terminating sensitization, 0. 2 ml of cell suspencion was tranferred from each culture flask into wells of the microtest plate. Triplicate samples were made and labelled with 3H-thymidine (3H-TdR, 0. 5 p,Ci/well, specific activity 5 Ci/mmole) for 18 hours, and the cells were harvested. The remaining MLC cells in the flasks were washed with Eagle's balanced salt solution and resuspended in CMC medium (consisting of MEM+ 10 % prescreened heat-inactivated fetal calf serum (FCS), 1% P /S, 1% NEAA, and 1% Lglutamine) for CMC assay. Freezing and thawing MLC cells. The cryopreservation method used was a slight modification of Miller et al.17). The cells were centrifuged and resuspended in the CMC medium, to which, then, were added an equal amount of cold-freezing medium containing 50% heat-inactivated FCS, 20% dimethyl sulfoxid_e (DMSO) in MEM to secure a final concentration of 30 % FCS, 10 % DMSO, and 10-20x106 cells/ml. One ml of this in a vial was kept in ice water for 5-10 minutes and placed in a -80°C freezer overnight prior to transferring into liquid nitrogen. The rate of freezing in this system is four times as fast as that in a rate-controlled freezer. For recovery of frozen cells, the vials were Generation of CTL to Human Monolayer Target Cells 51 thawed in 37°C water bath with gentle agitation until the last ice crystals disappeared. Thawed vials were kept in ice water and within five minutes the contents were slowly diluted with an equal volume of cold medium containing 15 % FCS and thoroughly mixed. This was repeated two more times to secure a final dilution of 1 : 8-1 : 12. This suspension was washed twice, and the pellets were resuspended in CMC medium for determination of viability, recovery, and cytotoxic activity. The persent viability of frozen-thawed cells was 86. 5±8.1% in unsensitized cells and 84. 6 ± 6. 7 % in sensitized cells, and the percent recovery was 53. 6±14. 7% in unsensitized cells and 62. 4±14. 1 % in sensitized cells. Cell culture. Culture of normal skin fibroblasts was initiated with finely miced explants from seven healthy donors. The culture was maintained in a complete medium consisting of 1% NEAA, 2 mM fresh L-glutamine, 1% P/S, and 15 % prescreened FCS in MEM. These fibroblasts were used as monolayer culture target cells in microcytotoxicity assay and as a source of HLA-SD antigens in IVS between the 2nd to 17th passages in vitro. 3 H-Proline cell-mediated cytotoxicity microassay. Cultured skin fibroblasts were labelled overnight with 50 μCi/ml of 3H-proline (specific activity 21. 9-24. 6 Ci/mmole), trypsinized, resuspended in test medium, and distributed into microtest plates at 1, 000 viable labelled target cells per well, as described elsewhere, 3>. Unless otherwise noted, a ratio of 250 effector cells to one target cell in used for 3 H-prolioe CMC assay. After 40 hours of incuaation in 5 % C02 and humidified air at 37° C, unattached dead target cells and effector cdls were washed away with 37°C phosphate-buffered saline containing 5% FCS. Then, the residual radioactivity of cells attached to each well was measured to indicate the number of viable target cells remaining. Analysis. Percent CMC (% reduction of target cells) is calculated as [1-(b)/(a)] x 100, where (a) =mean count per minute (com) of target monolayer incubated with effector cells from unsensitized culture, and (b) =mean cpm of target monolayer incubated with effector cells from sensitized culture. Here, for positive CTX of effector cells, more than 30 % reduction of target cells and significant difference (at P< 0. 05 by t-test) from mean cpm of five replicates of target monolayer incubated with medium alone were used. For the determination of specific CTX by effector cells from sensitization culture, negative CTX against control target and positive CTX against test target were considered.