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[CANCER RESEARCH 35, 2600-2608, September 1975] Evaluation of the Cell-mediated Immune Response to Murine Sarcoma Virus by [‘251]Iododeoxyuridine Assay and Comparison with Chromium 51 and Microcytotoxicity Assays Giuseppe Fossati,' Howard T. Holden,2 and Ronald B. Herberman3 Laboratory of Immunodiagnosis, National Cancer Institute, Bethesda, Maryland 200/4 the disease. Recently, detailed analyses of the primary cell mediated immune response to MSV have been performed The cell-mediated immune response of CS7BL/6 mice to by different laboratories (13, 14, 22, 23, 26). Nevertheless, murine sarcoma virus (MSV) was examined by the [125I]@ the results obtained were not concordant. With CRA, the iododeoxyuridine release cytotoxicity assay using MSV cell-mediated response appeared at 7 to 8 days after virus induced sarcoma tissue culture cell lines as target cells. inoculation, reached a peak at 13 to 14 days, and then Cellular cytotoxicity was detected as early as 3 days after declined rapidly (23, 26). In contrast, with visual MCA the virus inoculation. Most mice assayed between 12 and 17 cytotoxic activity of lymphoid cells was biphasic, showing days after MSV inoculation gave positive results with peaks in early and late periods after virus inoculation and maximum levels of activity present on Days 13 and 14. negative results at 13 to 14 days (22, 33). Because of these apparently discordant results, it was of interest to compare Reactivity was frequently detected for up to 100 days after MSV inoculation, although at low levels (5 to 10%). directly different assays for cellular immunity. A previous Additional experiments comparing the kinetics of the comparative study of the cellular immunity to a Gross virus-induced lymphoma in rats showed some correlation cellular response as measured by different in vitro cytotox icity assays were performed. The results showed a good between CRA and MCA, and many of the differences could direct correlation between the [‘25ljiododeoxyuridine re be accounted for by the different incubation periods in the lease assay and a 51Cr release assay. A similar pattern of assays (40). reactivity was also observed when the cellular response was In this study, in addition to CRA and MCA, the measured by a visual microcytotoxicity assay, although emphasis was placed on an assay in which cytotoxicity was reactivity dropped off more rapidly and became undetecta measured by the release of [‘251]IUdR. This assay has ble in most instances by 20 days after injection of MSV. previously been useful in detecting cell-mediated immunity Studies on effector cell type revealed that cytotoxicity in all in both experimental and human tumor systems (4, 30, 38). three assays was T-cell dependent, being eliminated by Like the MCA, it has a long incubation period and has been treatment with anti-O plus complement. Macrophages did thought to be a more objective and quantitative equivalent not appear to play a role, since treatment with carbonyl iron of MCA. and magnet had no effect. SUMMARY MATERIALS INTRODUCTION Injection of MSV4.into adult mice induces tumors at the site of inoculation, which undergo subsequent regression in most animals (1 1). By in vivo experiments, this regression has been shown to be mediated by immunological reactions (2, 3, 6—11, 24, 25, 32, 36, 37). These studies, however, ex amined only a few selected periods in the natural course of 1 Present Tumori, address: Instituto 20133 Milano, 2 Supported by NIH Nazionale per to Studio e Ia Cura dci Italy. Fellowship CA-5536l from the National Cancer Institute. S To 4 The whom requests abbreviations for reprints used are: should MSV, be sent. murine sarcoma virus; CRA, 51Cr release cytotoxicity assay; MCA, microcytotoxicity assay; [‘261]IUdR, [125I]iododeoxyuridmne; FBS, fetal bovine serum; PBS, 0.007 M phos phate-buffered solution of0.85% NaCI, pH 7.4. Received October 2600 9, 1974; accepted June 9, 1975. AND METHODS Animals. Male CS7BL/6N (hereafter called B6) mice, 3 to S months old, were obtained from the Rodent and Rabbit Production Section, Division of Research Services, NIH, and from the Mammalian Genetics and Animal Production Section of the Division of Cancer Treatment, National Cancer Institute, Bethesda, Md. Viruses. LMV-4 and DMV-6, 2 stocks of the Moloney strain of MSV (23), prepared from that originally obtained by Dr. A. Fefer from Dr. Moloney's laboratory (SVRP-l 14) were used. Tumor Cells. MSB and MSC tumor cells were estab lished tissue culture lines derived from MSV sarcomas induced in B6 and BALB/c mice, respectively. They were kindly provided by Dr. Gary Pearson, National Cancer Institute. Both of these cell lines release MSV and are tumorigemc in vivo (28, 32). CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research. VOL. 35 Ce//-mediated @ Immune Response to MS V thistocytoma were washed an additional time with PBS, combining P815 tumor cells were a transplantable DBA/2 mas theused with the cell pellet (“cells―). Samples containing and were grown in ascites form (5). The cells werefluid placedafter supernatant as well as those with cells were both immediately after removal from the animals andoriginal wasRBL-5 a well-type y scintillation counter, and each sample several passages in tissue culture.in (cpm).able cells were a Rauscher virus-induced transplant counted for 1 mm ofform, P815 cells, 10 cells were incubated in 1 ml lymphoma in B6 mice (29) and were grown in ascitesFor wereInoculations. with 1.0 @sCiof [‘26IJIUdRfor 6 hr. Then they as well as in tissue culture.medium athe Animals were inoculated i.m. with MSV inwashed 3 times, counted, and resuspended in medium at of l0@cells/mI. One-tenth ml of this suspen thigh. Detectable tumors at the site of inoculation wereconcentration attackerreached was seeded in each well and 0.5 to 1.0 x l0@ seen approximately 7 to 8 days after virus administration,sion wereregressed were added in 0.4 ml of medium. The plates their maximum size by 13 to 14 days, and thencells rockingage-matched at 37°in 5% CO2 for 16 to 20 hr on a by 18 to 20 days. Control animals were normal,incubated butgroup (the P815 cells did not become adherent mice receiving no inoculation. In addition, oneplatform fromcells, in suspension). The cells and supernatant received a single, i.p. injection of 3 x 10@P8 15 ascitesremained and the animals were tested 22 and 27 days later.such cells were harvested as above, except that no trypsini samplesthe Lymphocytes. Axillary, inguinal, and mesenteric (and inzation was needed for these suspension cells. The ofmice 1st set of experiments also cervical) lymph nodes fromwith P815 ascites cells were counted for 10 mm because in experimental and control groups were removedtheir [‘26I]IUdR.under low incorporation of as:ing sterile conditions and were placed in medium contain The percentage of cytotoxicity was calculated (NIHMedia Roswell Park Memorial Institute Medium 1640 % cytotoxicity IslandBiological Unit) with 20% heat-inactivated FBS (Grand (100IU/ml), Co., Grand Island, N. Y.), penicillin cpm in cells and streptomycin (100 @g/m1).The lymph nodes =100 x 100 were teased apart, and the cell suspension and fragmentswere cpm in cells + cpm in supernatant Aftercentrifugation, passed through a double cotton gauze sponge. the cells were resuspended in fresh mediumAll meansand tests were performed in quadruplicate, and the counted. Pooled lymphocytes of at least 2 immune miceand inwere standard errors of the percentage of cytotoxicity used in each test, and pooled lymphocytes from at leastthe calculated.2 experimental and the control groups were age-matched, syngeneic mice were used as controls.Experimental results were expressed as percentage of cel %manner cytotoxicity ± S.E. with the normal control Unless otherwise stated, spleen cells prepared in a similarlular subtracted out. Results were analyzed for were used in the CRA.cytotoxicity allAssays significance by Student's I test. In these experiments, cytotoxicity levels of 4% or more were significant for Cell-mediated Cytotoxicitycellular were[‘25I]IUdR at a level ofp < 0.05 and some values between 2 to 4% significant. In each test 4 replicates of target cells Release Assay. This test was done in Linbro with medium only were also included. The normal control FB-l6-24-TC tissue culture plates as previously described cytotoxicity in the experiments performed with MSB (27). From 10,000 to 20,000 MSC or MSB target cells in 1 cells averaged 16.0%, with a range of2.0 to 35.2%. ml of medium were seeded in each well, and the plates were CRA. This test was performed as previously described then incubated overnight at 37° in a moist 5% CO2 (23, 3 1). Briefly, 1 x l0@ spleen cells in Roswell Park atmosphere. The medium was then removed by aspiration,also Memorial Institute Medium 1640 with 20% FBS wereand 1 ml of fresh medium containing 0.5 ,tzCi of [‘251]IUdR with 5 x l0@ 5'Cr-labeled RBL-5 target cells (Amersham/Searle, Arlington Heights, Ill.) were added to (attacker:target cell ratio of 200:1) for 4 hr at 37°in a each well for 6 hr. The medium was then aspirated and CO2 atmosphere on a rocking platform. All tests excess radioactivity was removed by washing 3 times with were performed in quadruplicate. The amount of cell Hanks' balanced salt solution plus 10% FBS. Immediately lysis was determined by the cpm of 51Cr released into the after the last wash, lymphocytes were added to labeled calculatedthe supernatant fluid. The percentage of lysis was target cells at a 500 to 1000: 1 ratio in 1.5 ml of medium, andincubated as: lymphocyte:-target plates were incubated for 40 to 48 hr. The cell ratio was estimated according to the number of Lysis = cpm 51Cr released in experimental group < 100 target cells, which became adherent after plating, calculated cpm 51Cr released from cells by 4x freeze initially for each cell line in the 1st experiments. After. well,thawand incubation the supernatant was aspirated from each Thisfluid the attached cells were washed once with PBS. Controls for background release of 5'Cr for each experi cellswere was centrifuged and the supernatant and pelleted ment included incubation of labeled target cells with saved.Then PBSsolution 1.5 ml of EDTA-trypsin solution (Dulbecco autologous, unlabeled target cells. Experimental results were expressed as corrected percentage of cytotoxicity by EDTA)were containing 0.25% crude trypsin and 0.01% cellsuspension added to each well to detach the cells. The the subtraction of the background 5'Cr release. Differences between experimental and control groups were analyzed for was added to the 1st cell pellet, and the wells SEPTEMBER 1975 2601 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research. G. Fossati et a!. significance with Student's I test. A test was considered positive ifp < 0.05. MCA. The method used was basically that described by Hellström et a!. (15). Approximately 50 target cells (MSB) were seeded into each well of 96-well plastic Falcon Microtest II (No. 3040) culture plates. After overnight incubation, medium was discarded and lymphocytes were added in varying numbers. With this assay the attacker:tar get cell ratio was calculated on the basis of the number of target cells initially seeded. Eight replicates were used for each lymphocyte preparation. The plates were incubated at 370 in a 5% CO3 atmosphere for 40 to 48 hr. The wells were then washed carefully with PBS, and the cells remaining adherent to the plates were stained with Giemsa and counted under a microscope. Percentage of cytotoxicity was computed by the following formula: % cytotoxicity = NL —IL NL x 100 where NL is the mean of surviving target cells after incubation with normal lymphocytes and IL is the mean of surviving target cells after incubation with immune lympho cytes. Significance was evaluated by Student's t test. Treatmentof EffectorCells with Anti-OAntibody.AKR anti-U C3H antibody was produced by a procedure similar to that of Reif and Allen (35) that has been described in detail elsewhere ( 17). The main specificity of this reagent has been previously demonstrated (17). For further demon stration of the selective functional inactivation of T-cells with this reagent, B6 lymph node cells were treated with anti-O antibody plus complement and tested for proliferative response to mitogens. This treatment almost completely eliminated reactivity to phytohemagglutinin and con canavalin A (2 T-cell mitogens) while it did not affect stimulation by endotoxin, a B-cell mitogen (H. Kirchner and H. T. Holden, unpublished results). For determination of the effect of anti-O antibody on the cytotoxic response, lymph node cells were incubated for 30 mm at room temperature with a final dilution of 1:2 of antibody. The cells were washed once, resuspended in a 1:4 dilution of fresh rabbit serum as a source of complement, and then incubated at 37° for 45 mm. Controls were incubated at room temperature for 30 mm and then with rabbit serum at 37°for 45 mm. After 2 additional washings, the cells were counted, adjusted to the appropriate viable cell concentrations, and tested for residual cytotoxic activity in all 3 assays. Removal of Phagocytic Cells by the Iron-Magnet Tech nique.Phagocyticcells wereremovedfromthe lymphnode virtually eliminates suppressor activity (21) by macrophages. (20) and growth-inhibitory RESULTS Results of [‘25I}IUdR Release Assay after MSV Injec tion. In order to determine whether [‘25I]IUdR release assay was useful in detecting cell-mediated immunity in the mouse MSV system, we performed preliminary ex periments testing the cytotoxicity of lymphocytes from some MSV-infected animals, at different times after virus inoculation, against MSB and MSC target cells. As shown in Chart 1, cellular activity against MSB cells was de tected on Day 9, rose to a peak on Day 14, and then de dined on Day 16 and became negative by Day 24. The curve obtained with MSC cells had the same shape but lower cytotoxicity levels, indicating that this allogeneic cell line was less susceptible to lysis by immune lympho cytes from B6 animals. Subsequent experiments were performed with MSB cells, with a lymphocyte:target cell ratio ofSOO: I and with an incubation period of42 hr. Experiments were then performed to determine the influence of the time of incubation on the cellular cytotoxic ity results. When assays were terminated after 20 and 42 hr of incubation (Table I), considerably different results were seen. Cytotoxic activity was detectable at 20 hr but at low levels. The amount of cytotoxicity increased when the incubation time was extended to 42 hr. The level of detected activity was also affected by the lymphocyte:target cell ratios. As shown in Chart 2, percentage of cytotoxicity increased with the amount of lymphocytes added, both in the control and experimental groups. However, the percent age of experimental cytotoxicity above the normal control did not vary much, as the attacker:target cell ratio increased from 500 to 1500: 1. Subsequent experiments were per formed with a cell ratio of 500 to 1000: 1 and an incubation period of 40 to 48 hr. w C/, I1 CU 0 C0 C>. U 4 -4 preparations as previously described (20). Briefly, lymph w node cells were mixed with carbonyl iron (Technicon Instruments Corp., Tarrytown, N. Y.) and incubated at 37° for 30 mm. The tube containing the cell suspension was placed over a magnet and the cells in the supernatant, which 5 20 25 30 35 40 45 did not ingest the iron particles, were harvested with a DAYS AFTER VIRUS INOCULATION Pasteur pipet. Cells were washed and then used in the Chart 1. Kinetics ofcellular cytotoxicity in MSV-infected mice against assays. The carbonyl iron-treated cell suspension contained MSB (0) and MSC (•)target cells in the [‘°51]IUdR release assay. less than 1% macrophages (12). Furthermore, this technique Attacker:target cell ratio, 500: I. -4 -4 U 2602 CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research. @ I @ : @.- I@ Ce//-mediated Table I Etiect of incubation on cvtotoxic activity of l@'mphoci'tes from MS V-infected mice against MSB target cells Lymph node cells from mice inoculated with MSV were tested for their cytotoxicity against MSB cells in [‘°‘l]lUdR release assay with the dif ferent incubation periods. cellularcytotoxicity20 Days after virus incubation153.4 inoculation% 0.8aIS6.1 [email protected] a Mean ±SE. (p < 0.05). hr incubation42 hr ±0.5°5.5 ±[email protected]± ±0.2°9.2±0.2a Experimental value significantly ± above normal control - 03 40 x 0 - 30 - 20 - f--. 4 -4 -4 U 0 - 0— 0 250 500 000 LYMPHOCYTETARGET CELL RATtO On 2 occasions cytotoxicity 4 days of Cytotoxicity after virus inoculation. in [‘25I]IUdR Assay. Specificity studies were performed with P8 15 mastocytoma cells, since these cells have been shown to be good target cells for cellular cytotoxicity studies (I). Lymphocytes from MSV-infected mice and from B6 mice given injections of allogeneic P815 ascites cells were tested against both MSB and P81 5 ascites cells. P81 5-immune animals showed good activity against P8 15 ascites target cells, whereas no reactivity was detected against MSB cells (Table 2). Lymph node cells from MSV-immune mice, which were cytotoxic for MSB cells, had no killing effect against P815 ascites cells. @T@@oonegative results were obtained in [‘25I]IUdR releaseassay. Chart 2. Cytotoxic activity of lymphocytes from MSV-infected mice, 14 days after virus inoculation (S), and from normal mice (0) against MSB cells in the [1251]IUdR release assay. @ at 3 and in the [‘25I[IUdR release assay were also assayed at the same time in CRA and in MCA. Table 3 shows the results of the cellular cytotoxicity of MSV-infected mice and of mice inoculated with P815 ascites cells, measured by [‘25I]IUdR release assay and CRA against different target cells. In general, good correlation between the results of the 2 assays was observed. Qualitative concordance (statisti cally significant results in both tests) was found in 9 of I 3 tests performed. At high cytotoxic activity, there was also good quantitative concordance, except in 2 cases. However, when the level of cytotoxicity was low in CRA, lower or C0 C- @ Specificity cytotoxicity. early, Comparison of [‘25I]IUdRRelease Assay with Other Cytotoxicity Assays. Some of the mice tested for reactivity C- U detected to MS V Further experiments were performed with P8 15 target 60 — 50 was Response cells after several passages in tissue culture. A cross-reactiv ity was then observed. In fact, lymphocytes from MSV immune animals showed a higher percentage of cytotoxicity against P81 5 tissue culture cells than did lymphocytes from animals immunized against P815 ascites cells. Low, but significant, activity was detected by P815-immune mice on MSB cells. 70 — @ had no significant Immune The results of all these experiments are shown in Chart 3. Lymphocyte cytotoxicity of MSV-infected mice was tested at 28 different time points after virus inoculation starting on Day 3 and continuing over a period of 100 days. There was a considerable variability in cellular cytotoxicity ranging between —16.0 and 49.6%. For some of the time points after virus inoculation, repeated assays for cell-mediated cytotoxicity were performed. Here again there was a noticeable variability from one experiment to the next. At 14 days after MSV inoculation, 15 different experi ments were performed and the percentage of cytotoxicity observed varied between 3.7 and 49.6. In contrast, closer results were usually obtained on days where there was less cytotoxicity. Despite this variability in the results, there was a constant significant cytotoxicity of all animals tested between 12 and 17 days. After Day 21, until Day 100, low levels of cytotoxicity were often observed, although many animals SEPTEMBER 1975 Low-level reactivity persisted for over 8 weeks regardless of the assay used. Although the 2 assays as performed appeared to give 50T 4O@S >0 . 020— 0 .. .,O.— • -2O@@ 0 S -@—@__i@ 5 ‘0 5 20 DAYS 25 AFTER 30 ViRUS I 35 40 70 00 iNOCULATiON Chart 3. Cytotoxic activity of lymphocytes from MSV-infected mice tested against MSB target cells in the [‘26I]IUdRrelease assay at different days after virus inoculation. 2603 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research. G.Fossatieta!. Table 2 Specificity of cell-mediated cytotoxicitv in [‘251]IUdRrelease assay Lymph node cells from mice immunized with either MSV or P815 ascites cells were tested for their cytotoxic activity against MSB. P815 tissue culture, or P815 ascites cells. cytotoxicityP815mice after% Lymphocytes fromDays (ascites)MSV124.6 inoculated withinoculationMSB(tissue culture)P815 ±2.1MSV1528.6 1.1P8l5(ascites)27—5.4 [email protected] 0.8bP815(ascites)225.9 a Mean ±[email protected] ±1.6'—0.3 ±2.116.0 ±[email protected] ±1.0―18.2± ± ± ± l.4b ± SE. b Significant cytotoxicity above the control (p < 0.05). Table 3 Comparison of the [‘251]JUdRrelease assay with the CRA: cvtotoxicitv against different target cells B6 animals were immunized with MSV i.m. or P815 i.p. At different intervals after inoculation, lymphocytes were harvested and tested for cytotoxic activity against RBL-5, P815 tissue culture, or P815 ascites. cytotoxicity[‘25I]IUdR (ascites)[‘25l]IUdR I 5 (tissue culture)P8 Lymphocytesfrom inoculation% miceCRAMSV inoculated withDaysafter CRAP8 release assayRBL-5 ±l.l―@' MSV 27 1.3 ±0.7 @Ø4b 32.2 ±0.51 20.0±o5b 21.3±0.7b 11.7±0.2@@ 16.3±0.2@@ 7.4 ±0.2b 31.5±0.4@' 3.5±0.2' 25.5 ±0.5' MSV 48 5.7 ±0.6― 11.5 P815(ascites) P8l5(ascites)4 22 2713.1 4.6 ±2.5' MSV 12 MSV MSV 14 14 36.9 ±5.4@ 20.4 ±53I@ MSV 14 13.2±I.4b MSV 14 3.7 ±1.6 MSV MSV 16 21 34.0 ±0.9― —5.8±1.9 a Mean release assayCRA[‘25lJIUdR release assay ±2.1 ±0.8@ ±0.3' 2.6±0.3' @Ø3I@ 3.3±0.2b 18.2±1.4'22.3 19.7±0.2'1.4 16.0±1.2' 4.4 ±0.l@'26.3 5.9 ±1.0' —5.4 ±2.111.7 2.8±0.5k 41.2±0.3' 62.1±1.0' ± SE. b Experimental value significantly C Lymph from 2 animals nodes above pooled the for control [‘25I]IUdR (p < 0.05). release assay but correlative results, the [‘25I]IUdR release assay was per formed with lymph node cells, while the CRA used spleen cells as the effector population. Therefore we conducted a series of experiments in which lymph node cells and spleen cells were used in the CRA, while cells from the same lymph node poo1 were assayed in the [‘25IJIUdRrelease assay. As noted in Table 4, the activities of lymph node cells and spleen cells in the CRA at different times after injection of MSV were very close in most cases. Although quantitative differences were observed in some cases strong reactivity in the [‘25I]IUdR release assay was always accompanied by strong cytotoxicity in CRA, and weak [125IJIUdR release assay reactivity was accompanied by weak CRA reactivity, regardless of the source of lymphocytes. Table 5 shows the results in [‘25I]IUdRrelease assay and MCA at various times after MSV inoculation. Nine paired assays were performed, utilizing lymphocytes from the same pool of cells for a more direct comparison. Preliminary 2604 I5 spleens run individually in CRA. experiments with the MCA had shown that at a cell ratio of 1000: 1, no activity was found. However, by increasing the cell ratio to 2000 to 3000: 1, some significant activity was obtained. The general pattern of reactivity in the MCA was very similar to that observed in the CRA (Table 4; Ref. 23) and in the [‘25I]IUdR release assay (Chart 3). Significant cytotoxicity occurred during peak tumor growth, 10 to 15 days post-MSV injection, with a subsequent drop in activity by 20 days to nonsignificant levels. Occasionally, we observed positive reactivity at later time points (e.g., 31 days post-MSV in Table 5), but most mice tested after 20 days gave no significant cytotoxicity. The rapid drop in cytotoxicity at 20 days in the MCA was not seen in the [‘25I]IUdRrelease assay or in the CRA and may reflect the lower sensitivity of the MCA. Effect of Various Treatments on Cell-mediated Cytotoxic ity. Lymph node cells were taken from mice 13 days after MSV inoculation, treated with anti-O antibody plus comple CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research. II Cel/-mediated ment, and assayed for residual cytotoxicity in all 3 assays (Table 6). Treatment with anti-O resulted in almost complete loss of reactivity in the CRA, as previously reported (17), and had similar effects on cytotoxicity in the [‘25I]IUdR release assay and the MCA. As macrophages have been shown to exert a cytotoxic effect in some long-term assays, lymph node cells from MSV-immune animals at peak reactivity (13 days after inoculation) were treated with carbonyl iron and then tested for cytotoxic reactivity (Table 7). In all 3 assays there was no significant decrease in the cytotoxicity. In fact, in the are not Response to MS V IUdR release assay, were very similar to those reported earlier with CRA (23, 26). Reactivity usually appeared around Day 10 after MSV injection, with a sharp rise to peak cytotoxicity at 13 to 14 days. In 2 experiments, reactivity was also present at 3 and 4 days. Subsequently, the activity rapidly declined so that by Day 20 there was a low level of reactivity (4 to 8%), which persisted for over 100 days. Considerable variation was evident in the cytotoxic values reported for any one time point, e.g., on Day 14 the values [‘26IJIUdR releaseassay,therewasa significantincreasein the level of reactivity, indicating that macrophages required for the observed cytotoxic activity. Immune Comparison ofMCA Table 5 with [‘2'IIJL'dRrelease assay at various times at Eer inoculation with MS V Lymph node cells from mice immunized with MSV were tested at various times after immunization for their reactivity against MSB target cells in both the 1―IIIUdR relea@ assay and the MCA. Values for the 2 assays recorded on the same line were obtained with the same cell pools. DISCUSSION We have demonstrated that B6 mice inoculated with MSV develop cellular immunity against M SV-associated antigens, which can be measured by the release of [125I]@ lUdR from labeled MSV-transformed target cells. The kinetics of the immune response, as measured by [125I]@ release Days after MSV assay inoculationTumor 1000:1)2000:1―3000:15—@@I0.6c31.0—10.77—7.23.573d10+28.04à statusaMCA[‘25IJIUdR (500 Table 4 Cs'totoxicit, of li'mph node cells in the I 1251J1L!dRrelease assay and the CRA as compared to the reactivitt ofspleen cells in the CRA Pooled lymph node cells and/or pooled spleen cells from the same animal were tested at various times after immunization with MSV in the L2SIUdR release assay against MSB cells (lymph node cells only) and in the CRA against RBL-5 cells (spleen cells and lymph node cells). cytotoxicity[‘25l]lUdRrelease Daysafter assay in lymph MSV cells1323.7 inoculation% node cellsCRALymph node cellsSpleen ±0.2b15.8 0.4b1433.0± ±l.3a. b14.8 ±0.2'39Ø@Ø4b1533.9 1.6'55.1 0.3'20—3.8± ±0.9'33.3 ±Ø5b16.8 1.63.9±0.2―33@Ø3b218.7 0.2―22—5.7±±1.3'8.8 ±Ø3@)9.2 ±0.7'2714.8 1.010.4±0.6'5.8 0.5'348.9± ±0.4'9.0 ±2.5'7.8 l.5b6.8±0.4b6.4±0.4'36—3.1 ±0.5―4113.7±2.9―6.2±0.2b6.9±0.3― ±2.07.2±0.5―7.5 ± ± ± ± a Palpable tumor. +: b Lymphocyte:target a Mean ± SE. b Significant cytotoxic reactivity above the control (p < 0.05). C Percentage of d Significant cytotoxicity cytotoxicity no tumor, cell ratio. —. above above normal the control. control (p < 0.05). Table 6 Treatment of effector populations with anti-O antibody and complement Lymph node cells from mice given injections 13 days earlier with MSV were treated with anti-O antibody subsequently tested for residual cytotoxic activity. MCACRA(200:I―)[“IJIUdR assay(500-1000:1)2000:13000:1Complement alone40.5 2.2cAnti-O plus complement3. a Lymphocyte:target cell b Percentage of C Significant cytotoxicity SEPTEMBER cytotoxicity ±07@ C41.6 1 ±0.8c3.9 plus complement and release ±Ø3c20.4 ±0.5c6.9 ±5.6c36.0 ±5.5—4.0 ± ±2.8 ratio. above above the control control (p ± SE. < 0.05). 1975 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research. 2605 G.Fossatieta!. Table 7 oflvmph node allIs with iron and magnet Effect ofpretreatment Lymph node cells from mice injected 13 days earlier with MSV were treated with carbonyl iron and a magnet. The remaining cells, which did not ingest iron, were then tested for functional release CRA (200: 1)@[125IIIUdR assay (500- 1000: 1)MCA2000: 1Control37. I ±I .8―C42.3 l.2c51.0± [email protected]± a Lymphocyte:target cell b Percentage of cytotoxicity C Significant cytotoxicity ±0.4c22.6 1.0'19.7±7.7c39.8 13000: ±6.4e42.8 ± ratio. above above the control the control ± SE. (p < 0.05). ranged from 3.7 to 49.6%. At this time the source of such variation remains unknown. Some of the differences can probably be attributed to animal variation, whether due to different susceptibility to tumor growth, different cellular immune response, or slightly different amounts of virus in the inoculum. Variation in the cytotoxic reactivity of normal lymphocytes could also contribute to differences in test results, since experimental cytotoxic levels were relative to the base line produced by normal lymphocytes. We have observed normal reactivity in the CRA with RBL-5 target cells (17, 18) or rat lymphoma (C58NT)D target cells (18), and in the [‘25I]IUdRrelease assay, with human tumor cell lines (30), and similar observations have been made in other laboratories (19, 39). In addition, day-to-day differences in test conditions (e.g., medium, target cells) could be a source of variation in the observed cytotoxicity values. In the CRA we have been able to exclude variation due to normal lymphocyte reactivity by utilizing as a control unlabeled target cells in place of normal lymphocytes (23), and it has been possible to eliminate or monitor discrepancies due to fluctuations in test conditions by using frozen attacker and frozen target cells, which give consistent cytotoxicity values from day-to-day (H. T. Holden, R. K. Oldham, R. B. Herberman, and D. H. Lavrin, manuscript in preparation). With similar techniques, attempts are now being made to minimize variation in the [‘25I]IUdR release assay. Having established the kinetics of the immune response as measured by the [‘26I]IUdR release assay, it was of interest to compare these results to those obtained in the MCA and the CRA. Disparate results between the mi crocytotoxicity and 51Cr release assays had previously been reported by other laboratories. Cytotoxicity as measured by the CRA peaked at 13 to 14 days after MSV injection with a rapid decline to low levels by Days 20 to 30 in both BALB/c (22) and B6 animals (23, 26, 33). In contrast it has been reported that the cellular response after MSV injection as measured in the MCA, in both B6 and BALB/c animals, rose rapidly to a peak at 7 to 10 days [with some reports of activity as early as 1 day after inoculation (34)], dropped to low or undetectable levels by 12 days (peak tumor size), and then peaked again at 15 to 20 days with cytotoxicity levels remaining elevated for at least 30 days (22, 33). Several differences in the assay conditions could be cited to account for these discrepancies. The CRA used a 4-hr incubation period and suspension cells as targets. In contrast, the MCA utilized a 48-hr incubation period and adherent cells as 2606 activity. targets. Furthermore, the culture vessels were different, and many times the source of lymphocytes (spleen cells or lymph node cells) was not the same. These studies were initiated to understand better the relationship between the assays. To do this we tried to set up a radioisotopic assay that would measure a cell-mediated immune response to MSV and that would parallel the microcytotoxicity test but would be easier to quantitate. Therefore, attempts were made to establish assay conditions in the [‘251]IUdRrelease assay that were similar to those of MCA. Both assays used the same adherent target cells, similar incubation periods, a common source of attacker lymphocytes, and a close relationship in attacker:target cell ratios (500 to 1000:1 in [‘251]lUdR release assay 2000 to 3000:1 in MCA). This is in contrast to CRA where a com pletely different, nonadhrent cell was utilized as the tar get and there was a shorter incubation period, a lower attacker:target cell ratio, and usually a different source of attacker lymphocytes. In ourstudiesreportedhere,similarresultswereobtained in all 3 assays, with peak reactivity occurring at 14 days post-MSV injection and a subsequent decrease to low levels thereafter. Low-level activity persisted in the CRA and [‘25I]IUdR releaseassaybut not in the MCA, reflecting perhaps a difference in sensitivity of the assays. The [125I]IUdR release assay compared favorably to both the MCA and the CRA with regard to time of appearance of activity and pattern ofreactivity. In addition, the MCA and the CRA correlated closely. In contrast to previous reports (22, 23), activity was found in the MCA at the time of peak tumor size, while little cytotoxicity was detected beyond 20 to 21 days post-MSV. Unfortunately, the paired MCA and [‘25I]IUdR release assays (Table 5) did not always show correlative results, and the reason for this is unclear. One explanation might be that variation in assay conditions were responsible for the differences. Although the assays were set up under very similar conditions, there are intrinsic differences that could contribute to variations in one assay and not in another. Until assay conditions can be controlled or standardized to assure uniformity, such discrepancies will probably con tinue to be a problem. Not only were the patterns of reactivity similar in these cytotoxicity tests, but the effector cells appeared to be similar. Cytotoxic reactivity in all 3 assays was T-cell dependent, as it was removed after treatment of the effector CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research. Cell-mediated cells with anti-O antibody and complement, but not depend ent on macrophages, being unaffected by treatment by the iron-magnet technique. These finidngs are different from those of Plata et a!. (33 and Lamon et a!. (22) in the MCA and from those of Seeger et a!. (38) in the [‘25IJIUdR release assay. Previously, T-cells and non-T-cells were found to contribute to both peaks of cytotoxicity in the MCA, and the subsequent, persistent reactivity was as cribed to non-T-cells (22, 23). Similarily, non-T-cell killing was found by Seegar et a!. (38) in the [‘25IJIUdR release assay, although this activity was attributed to adherent cells, whereas the cytotoxicity in the MCA was thought to be mediated by B-cells (22, 23). The reasons for these major differences from previous reports are not clear at this time. Many differences in the experimental protocols can be cited. (a) Different virus preparations were used; however, they were all derived from a common source. (b) Although some workers used BALB/c animals for their investigations our studies were performed with B6 animals. However, Plata et a!. (33) also used B6 mice, and strain differences of the effector cells therefore were not a major factor. (c) The MCA can mea sure cytolysis as well as cytostasis, while the CRA and measure only cytolysis. In this study, the pattern of reactivity as measured by the MCA was similar to that of the CRA and the [‘25I]IUdR release assay, suggesting that cytolysis was the main effect we measured by the MCA. It is possible that, in other laboratories, cytostasis may be a more important factor in MCA results. (d) One parameter of the assays that was distinct from one laboratory to another was the target cells used. Although the cells were transformed by similar viruses (murine leukemia virus or MSV), it cannot be of the observed reactivity was against induced by the transforming agent. specificity are becoming increasingly as we become more aware of the concluded that all the same antigens Determinations of difficult to make, complex variety of sur face antigens that tumor cells possess. As shown by Her berman et a!. (16) with the CRA, spleen cells from MSV injected B6 animals were specifically reactive against tumor cells containing mouse endogenous virus-associated surface antigen and not (as would be expected) against tumor cells containing only MSV or murine leukemia virus antigens. The concordant results that we obtained in the 3 different assays suggest but do not prove that the antigens detected by all the tests were the same. Additional testing against other target cells would be necessary to answer this question. With regard to the last possibility discussed above, we have performed some tests that address themselves to the question of specificity. The MSB target cell used in the [ 125I]IUdR release assay was a syngeneic MSV-trans formed cell line. Initial testing indicated that this cell line was a more suitable target than the allogeneic line, MSC. The MSB line might be more susceptible to lysis than the MSC cells [although Seeger et a!. (38) success fully utilized MSC in a similar assay] or alternately B6 im munized animals may have recognized antigens on the MSB cell that were not found on the allogeneic cell line. Evidence supporting this difference in antigens on MSB SEPTEMBER 1975 Immune Response to MS V and MSC has been obtained in the CRA (16). Specificity was also tested utilizing an unrelated target cell. Lympho cytes from animals given injections of MSV 14 days earlier failed to kill [‘25I]IUdR-labeled P815 ascites cells. Similar results were obtained in the CRA. Con versely, in both the CRA and the [‘25I]IUdR release assay lymphocytes from P815-immunized animals had strong reactivity reactivity on MSB on P815 ascites cells and only a slight targets. As mentioned above, MSV spleen cells are cytotoxic in the CRA for cells carrying mouse endogenous virus associated antigen. P81 5 cells express this antigen after several passages in vitro and are then sensitive to the cytotoxic action of MSV lymphocytes (16). When the P815 tissue culture cells were used as a target in [1251]IUdR release assay with lymphocytes from 14-day MSV animals, they were positive while P815 ascites cells were not. This parallel testing, shows that the 2 assays could be measuring reactivity against common antigens related to mouse endog enous type C virus expression. However, we cannot con dude that this is the only reactivity that is being detected, since reactivity against more than I antigenic specificity could be generated by the inoculation of MSV into mice. 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