Colony Inhibition of Fibroblasts from Chimeric Dogs Mediated by the Dogs' Own Lymphocytes and Specifically Abrogated by their Serum

Colony Inhibition of Fibroblasts from Chimeric Dogs Mediated by the Dogs' OwnLymphocytes and Specifically Abrogated by their SerumAuthor(s): I. Hellström, K. E. Hellström, R. Storb and E. D. ThomasSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 66, No. 1 (May 15, 1970), pp. 65-71Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/60185 .

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ProceedinJ8 of the National Academy of Sciences Vol. 66, No. 1, pp. 65-71, May 1970

Colony Inhibition of Fibroblasts from Chimeric Dogs Mediated by the Dogs' Own Lymphocytes and Specifically

Abrogated by Their Serum

I. Hellstriorm, K. E. Hellstrom, R. Storb, and E. D. Thomas DEPARTMENTS OF MICROBIOLOGY, PATHOLOGY, AND MEDICINE, UNIVERSITY OF WASHINGTON

MEDICAL SCHOOL, SEATTLE

Communicated by R. D. Owen, January 16, 1970

Abstract. Nine canine irradiation chimeras were studied between 173 days and 7.5 years after 1200 to 1500 r of total body irradiation and transplantation of allogeneic bone marrow. Skin fibroblasts from chimeras and normal dogs were tested for colony inhibition by exposuie to sera and peripheral blood lymphocytes from both chimeric and normal dogs. Lymphocytes from the chimeric dog were found to inhibit colony formation by its "own" fibroblasts while lymphocytes from other chimeras or from normal dogs did not. Serum from the chimera specifically abrogated this inhibitory effect. These results indicate that the immunological "tolerance" of the chimeric dog is mediated in vivo by blocking serum factors.

Chimeras have been described to occur naturally in many animal species.1 Owen in 1945 described chimeric cows and showed them to contain two hemato- poietic cell populations, one of which was derived from the chimeric individual's nonidentical co-twin.2 A cow containing such a foreign cell population was found to accept skin grafts from the other twin permanently,3 a finding which initiated research leading to the concept of immunological tolerance.4

It is possible to induce chimeras experimentally by suppressing an animal's immunological response and inoculating it with allogeneic bone marrow. The first studies on chimera induction were performed in rodents.5 Subsequently, it was possible to establish permanent chimeras in dogs following supralethal whole body irradiation and transplantation of allogeneic bone marrow.6 The presence of foreign cells within the chimeric individual could be confirmed with cytogenetic techniques since female bone marrow was transplanted to males.'

It is not known why, in a permanent chimera, the immunologically competent foreign graft does not mount any harmful immunological reaction against its host. The most common explanation is that those lymphoid cell clones that would have been reactive against the foreign alloantigens of the host have been made "tolerant," i.e., specifically nonreactive.81-3 Alternatively, it has been suggested that a phenomenon analogous to immunological enhancement may be involved, by which a chimeric individual's serum can protect against such cellular immune reactions that would -otherwise lead to destruction of its own cells. 14-16

65



66 MEDICAL SCIENCES: HELLSTROM ET AL. PROC. N. A. S.

Hellstr6m et al. have employed an in vitro test, colony inhibition, to demon- strate specific cellular immunity to tumor antigens.'7 18 Inhibition of colony formation of plated target tumor cells was seen in both animal and human sys- tems after exposure of the target cells to lymphocytes from animals or human patients with tumors.'8-20 The degree of inhibition obtained was approximately the same regardless of whether the tumors had been removed or were actively growing at the time of the test. The seemingly paradoxical coexistence of cellular immunity detectable by an in vitro test, and progressive tumor growth in vivo, may be resolved by the demonstration that sera from tumor bearing animals (patients) contain factors, probably antibodies, which in a specific way protect the tumor cells from in vitro inhibition by immune lymphocytes.21 A similar mechanism may also operate under normal conditions. Lymphocytes from BALB/c female mice, pregnant with C3H males, can inhibit colony formation of plated C3H embryonic cells, indicating the presence of cellular immunity to paternally derived antigens in the embryos. However, the lymphocyte re- activity is nullified by blocking serum factors (antibodies?) which are present in the mothers' serum and are capable of specifically protecting plated embryonic cells from inhibition by immune lymphocytes.22

This paper describes experiments with dogs which lived through the period of greatest risk of secondary disease. They were in good health and were regarded as being permanent chimeras. Their lymphocyte populations were antigenically foreign to their hosts and, therefore, theoretically capable of mounting graft versus host reactions. Our aim was to study whether lymphocytes from such dogs were immune to antigens present in their explanted skin fibroblasts and whether serum from the same dogs contained any blocking factors capable of protecting the target cells against the lymphocyte effect.

Nine chimeric dogs were studied, numbered 258, 739, 483, 648, 655, 719, 728, 755, and 977.- All of them had been exposed to whole body irradiation followed by transplantation of allogeneic bone marrow cells.7' 232- Table 1 gives data on these dogs. Repopulation of the marrow by donor type cells was verified in six of these dogs. In the other three dogs, no marker existed for the verification of chimerism. However, in nine similar canine radiation chimeras with a sex difference between donor and host, cytogenetic studies demonstrated only donor type cells in the peripheral blood and bone marrow. Host type cells have never been found, even after intervals as long as five years.7' 24-27

Skin biopsies were performed and the tissue explanted in vitro. Fibroblasts growing out from trypsinized skin as well as from explanted pieces were used as target cells for colony inhibition tests. Peripheral blood lymphocytes were separated by a technique modified from one used with human material.20 From each lymphocyte donor, 50 ml blood was drawn into a syringe that contained 0.5 ml of a heparin solution (1000 units/ml) and mixed with 8 ml of plasmagel. The mixture was incubated for 20 to 30 minutes at 37?C. Cell-rich supernatant was then removed, centrifuged for seven minutes at 30 g to remove excess red cells, and transferred into 32 oz glass prescription bottles, to which 0.1 ml heparin solution/10 ml suspension was added. The bottles were incubated at 37?C for one hour, during which time most granulocytes and monocytes stuck to the



VOL. 66, 1970 MEDICAL SCIENCES: HELLSTR6M ET AL. 67

TABLE 1. Data on chimeric dogs. No. of marrow

Dog Irradi- Source of cells infused Evidence of Date of Date of colony no. ation (r) marrow (X 109) chimerism* grafting inhibition testing 258 1500 Sibling 10.5 No marker 2/2/67 10/13/69, 10/31/69 655 1500 Sibling 18.0 All donor cells by 2/17/69 11/5/69

cytogenetic analysis

719 1500 Sibling 30.4 No marker 2/14/69 11/5/69 728 1500 Sibling 7.1 All donor cells by 2/19/69 11/14/69

cytogenetic analysis

483 1200$ Sibling 16.5t No marker 8/14/68 10/27/69 739 1200t Unrelated 12.0 rbc's of donor type 3/4/69 10/13/69, 10/31/69 648 1200t Unrelated 20.6t rbc's of donor type 11/22/68 10/27/69 755 1490$ Unrelated 11.0 All donor cells by 4/25/62 2/13/70

cytogenetic analysis 977 1496$ Unrelated 44.8 All donor cells by 3/1/63 2/13/70

cytogenetic analysis * All dogs showed prompt hematological recovery, which is evidence of engraftment. t Marrow had been frozen to -800C in 10% DMSO. $ Methotrexate was administered after the marrow graft.

glass surfaces. The cells remaining in the supernatant were collected by centrif- ugation at 200 g for 20 minute and washed. This procedure yielded suspensions with about 80 per cent lymphocytes. Serum samples were harvested from the lymphocyte donors as well as from some other untreated dogs.

Colony inhibition tests were performed as in previous experiments with animal and human tumors.'7-2' Primary skin fibroblast cultures were trypsinized and the cells suspended in Waymouth's culture medium supplemented with nonessential amino acids, sodium pyruvate, L-glutamine and 30 per cent heat inactivated newborn calf serum. 50 mm Falcon plastic tissue culture dishes were seeded with 800 to 1000 cells each. The following day, when the target cells had attached to the Petri dishes, the culture medium was removed and 0.5 ml of the serum was added to each dish after dilution 1:5 in Eagle's minimum essential culture medium (MEM), supplemented with nonessential amino acids, sodium pyruvate and L-glutamine (MEM-suppl), immediately after which each dish received 0.5 ml of a leukocyte suspension containing 107 viable cells/ml, at least 80 per cent of which were lymphocytes. After 45 minutes incubation, 1.5 ml MEM-suppl was added to each dish, and the following day the dishes received an additional 1.5 ml MEM-suppl containing 20 per cent heat inactivated newborn calf serum. The dishes were incubated for three days, stained, the number of colonies counted and the differences in colony numbers calculated between experimental and control groups. Comparisons were made between the percentage colony inhibition obtained with a chimeric animal's own lymphocytes in the presence of either its own or control serum. The degree of protection by the dog's own serum is expressed in percent (100% protection = no inhibition).

A total of 12 experiments have been carried out, all of which are included in Table 2. It appears that lymphocytes from the nine chimeric dogs were significantly more inhibitory to fibroblasts from the same dogs than were lymphocytes from any of the controls, the degree of inhibition ranging between 25



TABLE 2. Data frm colony inhibition tests with explanted skin fibroblasts from chimeric dogs, exposed to peripheral blood lymphocytes and sera from the same chimeric dogs and from control animals.*

Percentage protection

Percentage with Target Lympho- No. of colonies colony autologous

Expt. cells cytes Serum (= SE) inhibition serum 1 258C 258C 258C 17.0 ?: 3.0 27.7 55.8

7N 3.0 ? 0.0 82.4 8N 9.0?1 0.0 52.6

739C 8.0 ? 2.0 52.9 8N 258C 23.5 -:? 6.5

7N 17.0? 1.0 8N 19.0 ? 1.0

739C 17.0? 1.0 2 739C 739C 739C 17.0: 1. 2 15.0 70.1

7N 10.0 ? 0.0 47.3 258C 10.3 ? 0.9 53.2

7N 739C 20.0 ? 2.0 7N 19.0?h 1.0

258C 22.0 ? 1.0 3 648C 648C 648C 38.0 ? 1.5 14.8 68.4

483C 26 44.9 840N 24 40.7 839N 24 53.4

840N 648C 41.0 ? 1.0 483C 46 840N 37 839N 46

839N 648C 47.0 ? 4.0 483C 47.7 ?: 4.7 84ON 47.5 ?t 1.5 839N 53.3 ? 2.2

4 483C 483C 483C 56.7 ?L 2.4 -21.1 132.0 648C 14 75.4 840N 15 63.9 839N 23 58.0

840N 483C 42.0?4.0 648C 68 84ON 39 839N 59

839N 483C 51.5 ? 5.5 648C 53.3 ? 7.9 840N 45 839N 58.0?1.0

648C 839N 44 5 739C 739C 739C 17.9 ? 1.2 10.2 82.7

839N 9.5 ? 2.5 69.0 840N 11.0 ? 0.0 56.0 258C 12.5?:b 2.5 52.3

839C 7390 19.7 ? 1.2 839N 30.7 ? 2.2 840N 25. 0 3.5 258C 28.3 ?b 1.2

840C 7390 20.3 ? 1.2 839N 25.0 ?2 2.5 840N 25. 0 ?1: 2.5 258C 24.3 ?L 3.5

* C (following the number of the dog) = chimera; N = normal untreated dog. The target cells were exposed to sera from those chimeric dogs whose sin was explanted and to

control sera. Between 2 and 5 Petri dishes were included in each group. Percentage reduction of colony formation by lymphocytes from the chimeric donor of the target



TABLE 2 (continued) Percentage protection

Percentage with Target Lympho- No. of colonies colony autologou.s

Expt. celis cytes Serum (+ SE) inhibition serum 6 258C 258C 258C 60.3 + 1.3 -42.6 193.0

840N 29.3 +- 3.8 46.4 839N 27. 0 4t 1. 5 44.1 739C 27.3 4? 4.8 46.8

839C 258C 42.3 + 3.8 840N 54.7 +E 2.6 839N 48.3 +E 2.4 739C 51.342.7

840C 258C 44.0 + 0.0 840N 46. 0 + 3. 0 839N 42.5 4 1 .5 739C 47.0 + 5.0

7 728C 728C 729C 117.5 +- 5.5 4.9 84.3 840N 85.5 :+ 5.5 33.1 839N 83 .0 +t 13.0 29.4

839N 728C 123.5 + 5.5 840N 127.8 + 8.5 839N 117.5 ? 4.7

8 839N 839N 839N 57.3 +- 1.8 6.1 0 84ON 67.0 + 3.2 -4.7 728C 77.7 + 6.3 -5.0

728C 839N 61.0 840N 64.0 728C 74.0

9 719C 719C 7190 72.0+ 2.1 39.2 33.0 655C 100.3 +- 7.2 24.6 60.0

7N 40.7 + 0.3 61.4 8N 64.0+ 2.1 55.6

655N 719C 118.5 + 1.5 655C 133.0 + 15.0

7N 105.5 + 2.5 8N 144.0 +4 4.0

10 655C 655C 655C 100.3 ?E 7.2 30.8 35.5 7N 75.7 +t 6.4 45.3 8N 74.7 + 3.7 50.2

7190 86.3 + 9.5 40.3 15.6 719C 655C 134.5 + 5.5 7.2

7N 102.0+ 12.0 26.4 8N 138.0 + 6.0 8.0

719C 108.0 +t 12.0 25.3 8N 655C 145.0 + 7.0

7N 138.5 +E 5.5 8N 150.0

719C 144.5 + 0.5 11 755C 755C 755C 78.7 ?4 5.4 0.4 99.2

977C 44.7 + 6.4 52.9 840N 51.3 +t 4.4 46.6

840N 755C 79.0 + 5.0 977C 95.0 840N 96.0

12 977C 977C 977C 119.7 +- 3.8 25.6 39.2 839N 70.0 + 4.0 42.1

7550 977C 131.0 ? 1.0 839N 121.0

cells was determined by comparing colony numbers in plates receiving such lymphocytes with plates receiving control lymphocytes and the same serum, the latter values being pooled before the com- putation. S Sera from the donors of the target cells were then compared with control sera for their ability to abrogate the inhibitory effect of lymphocytes from the target cell donors; this ability is expressed in percent.



70 MEDICAL SCIENCES: HELLSTROM ET AL. PROC. N. A. S.

and 80 per cent. It also appears that this inhibition was specific. Lymphocytes inhibiting a chimeric dog's own fibroblasts did not inhibit such cells from other chimeric or normal dogs.

The colony inhibition obtained with lymphocytes from a chimeric dog was abrogated by serum from the same dog but not by serum from the control dogs. Serum protecting target cells from one dog did not protect cells from another, except in experiments 9 and 10, in which some cross-reactions were detected.

The present findings suggest that the immunological nonreactivity (tolerance) of the chimeric dogs in vivo is mediated by blocking serum factors. It may be speculated, on the basis of analogy with data from experiments on Moloney sarcomas in mice, that the blocking factors are antibodies,"9 but there is as yet no evidence that this is the case. Our results contradict the hypothesis that those lymphocyte clones that could react immunologically against a chimeric animal's own skin fibroblasts had been depleted or inactivated. These indicate why allogeneic inhibition does not occur in the chimeras studied, since allogeneic inhibition can be abrogated by serum from animals immune either against the target cells or against the foreign surface antigens of the lymphocytes.28 It remains to be studied whether blocking serum factors can be revealed also in naturally occurring chimeras, in animals made tolerant as newborns by inocula- tion of allogeneic cells, and in allophenic animals.29 The presence of such factors has clear implications for interpreting the phenomenon of immunological tolerance.

This work was supported by grants CA-10188, CA-10189, and AI-09419 from the National Institutes of Health, and by grants T453 and T-280 from the American Cancer Society. Dr. Thomas is the recipient of a Research Career Award, AI-2425, from the National Institute of Allergy and Infectious Disease. The skillful technical assistance of Mrs. Lydia Cabasco, Mr. T. Graham, Mrs. Ingalill Mosonov, Miss Karen Ritland, and Mrs. Violet Russell is gratefully acknowledged.

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(1951). 4Billingham, R. E., L. Brent, and P. B. Medawar, Nature, 172, 603 (1953). 5 Lindsley, D. L., T. T. Odell, and F. G. Tausche, Proc. Soc. Exptl. Biol. Med., 90,512 (1955). 6 Thomas, E. D., R. Storb, R. B. Epstein, and R. H. Rudolph, Transplantation Proceedings,

1, 31 (1969). 7 Epstein, R. B., J. Bryant, and E. D. Thomas, Transplantation, 5, 267 (1967). 8 van Bekkum, D. W., Ann. N.Y. Acad. Sci., 99, 550 (1962). 9 Burnet, M., "The Integrity of the Body," Howard Books in Biology (Cambridge: Harvard

University Press, 1962). 10 Wilson, D. B., and R. E. Billingham, Adv. Immunol., 7, 189 (1967). 11 Vos, O., and W. W. H. Weyzen, Transp. Bull., 30, 117 (1962). 12 Cole, L. J., and W. E. Davis, Jr., these PROCEEDINGS, 47, 594 (1961). 13 Prehn, R. T., and D. R. Thursh, in Mechanisms of Immunological Tolerance, ed. M. Hasek,

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VOL. 66, 1970 MEDICAL SCIENCES: HELLSTROM ET AL. 71

19 Hellstr6m, I., and K. E. Hellstr6m, Int. J. Cancer, 4, 587 (1969). 20 Hellstrom, I., K. E. Hellstr6m, G. E. Pierce, and J. P. S. Yang, Nature, 220, 1352 (1968). 21 Hellstrom, I., K. E. Hellstrom, C. A. Evans, G. H. Heppner, G. E. Pierce, and J. P. S.

Yang, these PROCEEDINGS, 62, 362 (1969). 22 Hellstrom, K. E., I. Hellstr6m, and J. Brawn, Nature, 224, 914 (1969). 23 Epstein, R. B., R. Storb, H. Ragde, and E. D. Thomas, Transplantation, 6, 45 (1968). 24 Storb, R., R. B. Epstein, R. F. LeBlond, R. H. Rudolph, and E. D. Thomas, Blood, 33,

918 (1969). 21 Epstein, R. B., R. Storb, R. A. Clift, and E. D. Thomas, Transplantation, 8, 496 (1969). 26 Storb, R., R. B. Epstein, T. C. Graham, and E. D. Thomas, Transplantation, 9, 240 (1970). 27 Storb, R., R. B. Epstein, J. Bryant, H. Ragde, and E. D. Thomas, Transplantation, 6,

587 (1968). 28 Hellstr6m, K. E., and I. Hellstr6m, Progr. Exptl. Tumor Res., 9, 40 (1967). 29 Mintz, B., and W. K. Silvers, Science, 158, 1428 (1967).



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Colony Inhibition of Fibroblasts from Chimeric Dogs Mediated by the Dogs' Own Lymphocytes and Specifically Abrogated by their Serum