Br;ef Commun;caf;on: Cell-Mediated Immunity in Mice Against Papain-Solubilized Histocompatibility and Tumor-Specific Antigens by a Macrophage Migration Inhibition Microassay 1 B. A. Maurer,2 J. H. Dean,> J. L. McCoy,2 E. Appella,3 and L. W. Law 3, SUMMARY-The cell-mediated immune status of B10.02 (H-2 d ) mice immunized. with spleen cells from a congenic strain, B10.A (H·2 a ) , differing at the H-2 locus and of BALB/c mice immunized with a syngeneic simian virus 40 (SV40)induced sarcoma (mKSA-TU5) was evaluated by an agarose microassay for migration inhibition factor. The inducing antigens in this experiment were papain-solubilized and partially purified chromatographie preparations of spleen cells from AIJ mice (H-2 a) and a papain-solubilized antigen extract prepared from a tissue culture-adapted cell line (TU-5), derived from the SV4Q-induced mKSA tumor. The assay used microliters of normal or immune peritoneal exudate cells (PEC) resuspended in a 2-pol droplet of agarose and cultured in the presence or absence of antigen. Specific migration inhibition of PEC from immunized mice was observed with concentrations of solubilized antigen preparations as low as 2.0 pogl ml (0.67 j.tgl chamber).-J Hatl Cancer Inst 56: 1075-1078, 1976.

Papain-solubilized and partially purified antigen preparations of mouse H-2 histocompatibility antigens and simian virus 40 (SV40) tumor-specific antigens (TSA) (1~ 2) have been partially characterized and shown to retain the capacity to induce specific skin graft rejection, immunologie enhancement, tolerance in the case of the H-2 antigens (3-5), and specific transplantation rejection of syngeneic SV40 tumor cells (1, 2) in the case of TSA. We determined whether these antigen preparations, i.e., H-2 a and SV40 TSA, could be used to detect a cellmediated immune (CMI) response in immunized miee with an in vitro CMI correlate. The mieroassay for migration inhibition factor (MIF) which uses agarose droplets, first described by Harrington and Stastny (6) and developed in this laboratory with peritoneal exudate cells (PEC) from individual miee (7) was utilized. MATERIALS ANO METHOOS

Mouse strains.-BALBjc mice were supplied from the breeding colony of the NCI. The BIO.D2, BIO.A, and C57BLjScSn(BIO) congenic strains of mice were obtained from the Laboratory of Cell Biology, NCI. These latter . mice differ at the strong H-2 locus. Brother X sister inbreeding was continued from a nucleus of mice of each strain obtained from Dr. jack Stimßing, McLaughlin Research Institute, Great Falls, Montana, and Dr. George Snell, The Jackson Laboratory, Bar Harbor, Maine. Antigens.-The H-2 antigen preparation was obtained from the membranes of Aj J spleen cells as described in (5). Limited papain digestion followed by G-150 Sepha'dex chromatography yielded FI, F2, and F3 fractions of which only the F2 fraction has specific biologie activity including skin graft rejection, as detected in congenic mice (3-5). The partially purified F2 fractions of spleen cells of the H-Za haplotype mice used were also capable of inhibiting 51Cr release by antisera made against the determinants H-2.4, H-2.5, H-2.23, and H-2.25 (5).


The antigen preparation from membranes of dissociated tissue culture-adapted TU-5 cells from the SV40 virus-induced mKSA tumor (8) was obtained by papain digestion as described in (1~ 2). TU-5 cells were adapted to tissue culture from the ascites mKSA fibrosarcoma and have undergone more than 100 passages in vi tro. The crude membrane, crude soluble (CS), and the F2 and F3 G-150 Sephadex fractions all provided specific resistance to transplantation of syngeneic mKSA tumor cells (1~ 2). The CS antigen preparation used in this study was assayed for in vivo immunogenicity as discussed later. Animal immunization.-BIO.D2 (H-2d) mice were immunized to H-2 alloantigens by three weekly injections of 60 X 106 BIO.A (H-2 a ) or BIO (H-2 b) spleen cells. These animals were given a fourth injection of spleen cells approximately I month after the last weekly injection. This immunization protocol was based on that used for obtaining maximum inhibition by purified protein derivative (PPD) of PEC from BCG-immunized mice (7). BALB j c mice were also made immune to mKSA, which had been shown to possess a strong tumor-specific transplantation antigen (1~ 8), by four weekly sc injections of 105 viable, tissue culture-grown mKSA-TU5 cells. Imrnunity ' was confirmed with representative mice from each immune group by sc challenge with I X 106 mKSA asci tes cells. Mice were made immune to KA-3l (9) tumor challenge by six weekly ip injections of 2-15 X 1()6 nonproducer KA-31 cells irradiated with 15,000 R. We confirmed immunity in syngeneic BALBjc mice immunized with X-irradiated tumor cells by challenging control and experimental groups with graded doses of viable syngeneic KA-31 cells. Agarose drop let assay [or MIF.-The assay was patterned after that described by Harrington and Stastny (6) and was developed with mouse PEC by Maurer et al. (7). In general, the procedure involves the placement of a 2-,ul droplet containing 5-6 X 105 PEC in 0.2% agarose (Marine Colloids, Rockland, Me.) containing TC 199 and 15% fetal calf serum with or without antigen. After 24 hours' incubation at 37° C, the area of PEC migration was determined by planimetry and the percent inhibition was determined by com parison of the mean area of migration of four replicates with antigen to that without antigen. We determined the statistieal significance by using Student's t-test to compare the control and test means. We originally standardized the assay in our laboratory by using soluble PPD antigen and normal PEC or PEC from mice immunized with BCG. Received September 23, 1975; accepted November 25, 1975. Litton Bionetics, Department of Immunology, 5516 Nicholson Lane, Kensington, Md. 20795. 3 Laboratory of Cell Biology, National Cancer Instiute (NCI), National Institutes of Health, Public Health Service, V.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 4 We acknowledge the technicaI assistance of Joyce Williams, Lawrence jerome, and David Siwarski. 1






RESULTS Migration Inhibition with H-2a Antigens

PEC were harvested from nonimmunized BIO.D2 (H-2 d ) and BIO.D2 mice immunized with spleen cells from BIOA (H-2 a ) mice. The active (F2) fraction (5) of papainsolubilized and chromatographically purified H-2 a antigen was used in these experiments. The results of one of four representative experiments, all of wh ich gave positive inhibition results, are presented in table 1. PEC from immunized BIO.D2 mice were inhibited by soluble H-2 a antigen in concentrations of 2.0-200 ftgjml. The inhibition of migration of PEC with antigen levels up to 200 ftgjml was not due to toxicity, since this concentration of l.-Inhibition of PEC migrationfrom Bl0.D2 mice immunized with BtO.A epleen cells with the use of soluble, partially purified, H-2" histocompatibility antigen


N onimmunized mice Antigen - - - - - - - - - - Average ",g/ml a Percent migration, cm- inhibition (±SE) 48.1 52.2 59.5 57.6 48.0 52.5

0 2.0 8.0 25.0 100.0 200.0

(±2.7) (±0.66) (±1.4) (±0.66) (±1.3) (±4.9)

0 +8.5 +23.7 +19.7 0.3 +9.1


Immunized mice Migration, cm? (±SE) 42.9 33.9 31.5 34.4 29.0 21.1

Percent inhibition

Migration Inhibition with SV40 Tumor Antigen(s)

(±0.95) (±1.7) (±1.8) (±0.7) (±1.6) (±1.3)

0 21.0 26.6 19.9 32.5 50.9

c c c

d d

a H-2a antigen preparation from A/J spleen cells. b + indicates migrat.ion greater than control migration. , Mean differs sig niflcantly from control mean by P 0.01. d Mean differs aignificently from control mean by P

Cell-mediated immunity in mice against papain-solubilized histocompatibility and tumor-specific antigens by a macrophage migration inhibition microassay.

Br;ef Commun;caf;on: Cell-Mediated Immunity in Mice Against Papain-Solubilized Histocompatibility and Tumor-Specific Antigens by a Macrophage Migratio...
409KB Sizes 0 Downloads 0 Views