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Prevention of tumor growth in an “immunologically privileged site” by adoptive transfer of tumor-specific transplantation immunity
JOURNAL
OF
SURGICAL
RESE.4RCI1,
12,
62-69
(1972)
PREVENTION OF TUMOR GROWTH IN AN “IMMUNOLOGICALLY PRIVILEGED SITE” BY ADOPTIVE TRANSFER OF TUMORSPECIFIC TRANSPLANTATION IMMUNITY J. WILLIAM
FUTRELL,
M.D.,
NILE
L.
ALBRIGHT,
GEORGE
MATERIALS
H.
MYERS,
JR.,
M.D.
AND
filETHODS
Inbred female Sewall-Wright strain 2 guinea pigs, obtained from the Laboratory Branch of the National Institutes of Health, were used. These animals uniformly accept first and second set isograft,s. In each experiment,, all animals were age-matched and housed in group cages containing six animals. Water and Wayne guinea pig chow with supplemental ad libif~~~~~. A methylcarrots were available cholanthrene-induced liposarcomn, dcsignat,ed MCA-A, and shown to possess tumor-specific transplantation antigens [9], n-as used in all experiments. The tumor was inducaed by subcubaneous injection of 4.0 mg. ?IICd and has been maintained in our laboratory by periodic transplantation int.0 female strain 2 guinea pigs. All experiments employed tumor grafts earlier than the tenth transplant generation. Using our own modification of a procedure devised by Frey and Wenk [.5], and later adapted by Barker and Billingham [l], circular, full-thickness skin pedicles approximately 4 cm. in diameter and containing the panniculus carnosus muscle were cut’ from the shaved flanks of ether-anesthet’ized guinea pigs. A prominent neurovascular stalk supplying t,he isolated segment of flank skin, and comprised of a perforating intercost,al artery and vein with some adjacent neural fibers, n-as identi-
From The Surgery Branch, National Cancer InstiNational Institutes of Health, Bethesda, tute, Maryland. Submitted for publication November 19, 1971. 62 Q 1972 by Academic Press, Inc.
AND
elucidated, however. To this end a model was developed to study the effect of adoptive t,ransfer of tumor-specific transplantation immunity on the growth of a syngeneic tumor in a surgically created immunologically “privileged site” in in-bred guinea pigs.
EVIDENCE CONTINUES TO ACCUAIIJLATE that t,he development of human ncoplastic disease is in considerable measure relatcd to the degree of immunocompetence of the host organism [4]. It is now well established that in the presence of systemic immunodepression, whether from drugs, irradiation, or other causes, the liklihood of development and progression of cancer is considerably incrcascd [13]. Based upon experimental work demonstrating increased tumor growth in areas totally deprived of afferent lymphatic drainage, we have proposed that it is conceivable that neoplasia may grow preferentially in areas of “localized immunosuppression” in which a normally immunocompetent host is unable to recognize the foreign antigenicit,y of tumor cells [6, 71. A unique experimental model for studying the capability of an organism to immunologically recognize and reject genetically nonident,ical t(issue grafts has been the use of transplants to so-called immunologically privileged sites [I, 2, 141. Such arcas lack normal, funct,ional afferent, lymphatic drainage and are, by definition, anatomic sites within the host milieu where foreign tissue is protected against the normal immunologic rejection process and allowed a prolonged survival. Perhaps the best studied examples of such sites are the hamster check pouch [2] and the ant.erior chamber of the eye [12]. The role of “immunologically privileged sites” relative to t)umorspecific transplantation immunity has not been
Copyright
M.D.,
FUTRELL,
ALBRIGHT,
AND MYERS:
fied and isolated. Any adherent areolar tissue or connecting fibers was sharply excised. The resulting circular skin defect was then closed longitudinally with interrupted 3-O nylon sutures leaving a small cent,ral aperture for emergence of the vascular pedicle. A double sterile sheet of Adaptic nonadherent dressing (Johnson and Johnson, New Brunswick, KJ) with a cent#ral slit to accommodate t,he vascular stalk was placed over the wound and under the raw surface of the skin flap. The pedicle was t,hen repositioned on the flank and stabilized with surrounding adhesive tape. An additional sterile dressing of Telfa (Kendall, Chicago, IL) was applied with multiple strips of wat’errepellent adhesive tape. The dressing allowed normal mobilit,y and prot’ection of the pedicle from cannibalism. The adequacy of complete aff erent lymphat,ic int.erruption in this model has previously been reported [I, 61. A single-cell suspension of the MCA-A tumor was prepared by t,rypsin enzymatic dissociation of the solid tumor according to the technique of Madden and Burke [lo]. This process yielded a suspension cont.aining 90-95 % viable tumor cells as judged by trypan blue (0.05 70) exclusion. The cells were suspended in media RPM1 with 20 % fetal calf serum and the conc&rat,ion adjusted so that 0.05 ml contained the desired number of viable tumor cells. A Zgaugc needle 011 a tuberculin syringe was used for all inject,ions. Meticulous dressing daily and t,hereafter less changes, initially frequently, \vcrc necessary to assure good viability of the pedicle with no chance for reatt.achment.. Thrice weekly inspection and palpation of the pedirles was employed to dctermine the time of initial appearance of tumor nodules. After this, measurement of the two largest perpendicular diameters of the tumor was performed. After each inspection, careful redressing was performed. Additional normal strain 2 animals lvere injected weekly for (i-10 weeks with a subgrowth intradermal inoculum of 1 X lo6 NCA-A tumor cells for preparation of immune cells and serum for adoptive t,ransfer of tumorspecific transplantat,ion immunity. The animals were exsanguinated by intracardiac blood aspiration and serum was obtained by centrifugation of the whole blood at 200g for 10 minutes.
PREVENTION
OF TUMOR
G3
GROWTH
After t,his spleen cell suspensions were made from spleens aseptically excised from t,hese MCA-A immune strain 2 animals. The spleens were individually minced with scissors in Earle’s balanced salt solution and passed through 40-mesh stainless-steel screens. The suspension was then filtered through SO-mesh stainless&eel screens and centrifuged at. 2OOg for 10 minutes. The cell button was resuspended in media RPM1 1640 with 20% fetal calf serum. Viability was determined by trypan blue exclusion and the concentration adjust,ed to contain 1 X log viable spleen cells. Lymph node cells were prepared in a similar manner from the axillary and inguinal nodes of multiple RICh-A-immune animals. EXPERIRIE?;TS
ASD
RESULTS
Experiment 1. Precentiojl of “Prizdegecl Site” Tumor Growth by ;ldoptive Tratisjer ctf CellWe have previously shown that 1 X lo6 MCA-A tumor cells will grow progressively in a syngeneic host resulting in death of the animal when inoculated into an al~mphntio skin pediclc “privileged site” [7]. The same size tumor inoculum is routinely rejected n-hen given intradermall\r in an area with intact lymphatic drainage. In order to assess the effectiveness of passive adminisr rution of immune spleen cells in protecting ngainst such t,umor growth in an immunologically privileged site, the following experiment was performed. Alymphatic skin pcdicles were constructed 011 the flanks of nine animals and randomlg inoculated with 1 X lo6 viable syngencic MCA-A tumor cells (I’ig. 1). In addition, three animals received an intraperitoneal injection of 1 X log immune spleen cells from isologous animals previously rejecting multiple intradermal inocula of MCA-A tumor cells. Tllree animals received 1 X log nor1na.l spleen cells intraperitoneally from isologous animals while the remaining animals received no spleen cells. Large progressive tumors grew on the alymphatic pedicles of all animals receiving either no spleen cells (3/3) or normal isologous spleen cells (3/3) (Fig. 2). Two of the three animals receiving intraperitoneal immune isologous spleen cells were protected against “privileged
64
JOURNAL
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growth
OF
Tumor
SURGICAL
RESEARCH,
Tumor
Fig. 1. Diagrammatic scheme of Exp. 1 showing inoculation of 1 X lo6 MCA-A syngeneic t,umor cells into alymphatic skin pedicles of strain 2 guinea pigs. Those animals which, in addition, received an intraperitoneal injection of 1 X lo8 spleen cells from syngeneic animals previously rejecting the MCA-A tumor xere protected against “privileged site” tumor growth. Animals which received normal syngeneic spleen cells or no spleen cells grew large tumors.
site” kunors. The one t’umor that occurred in an animal receiving intraperitoneal immune spleen cells was significantly smaller t’han those of the controls (Fig. 3). Experiment 2. Failure of Immune Spleen or Lymph Node Cells to Confer Tumor-SpeciJic Immunity When Administered Introdermally or Subcutaneously to Nonimmune Animals Having established that spleen cells from MCA-A immune animals were capable of adoptive transfer of tumor-specific transplantation immunity to an immunologically “privileged site”, the following experiment was designed to determine the effect of the route of administration of the immune cells on the de-
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FEBRUARY
1972
gree of conferred immunity. Alymphatic skin pedicles were constructed on nine animals and inoculated with 1 X lo6 MCA-A tumor cells (Fig. 4). Three random animals also received an int,radermal/subcut’aneous inoculation of 1 X log immune isologous spleen cells, while I hree received 1 X log intradermal/subcut,aneous lymph node cells from MCA-A-immune isologous animals. The remaining animals received no immune cells. All animals (9/9) gre\\ large, progressive “privileged site” tumors. In an attempt to demonstrat,e some protcction against “privileged site” tumor growth by adoptive transfer of intradermally/subcutaneously administered immune spleen and lymph node cells, the above experiment leas repeated using one-half the number of tumor cells (5 X lo5 I\ICA-A) and multiple inoculations of the immune spleen and lymph node cells. The immune cells were harvested and administered on three separate occasionsonce 2 days prior to tumor inoculation and 1 and 4 days aft,er tumor inoculation. Again no tumor prevention was demonstrated, however, in that all animals grew large “privileged site” tumors (Fig. 5). Experiment S. The E$ect of Isologous LLImmune” Serum on MCA-il Tumor Growth Although cell-mediated immunity is thought to be the primary component in the immunologic rejection of chemically induced syngeneic tumor grafts, a series of animals was studied to define the effect of serum from animals previously rejecting the MCA-A tumor on syngeneic tumor growth in an immunologically “privileged site.” Alymphatic skin pedicles were constructed on eight animals and inoculated with 5 X lo5 MCA-A tumor cells. Eight additional animals received a normally subgrowth intradermal inoculum of 5 X IO5 MCA-A tumor cells in an area with intact lymphatics. Immune serum from isologous animals previously rejecting multiple intradermal MCA-A tumor inocula was administered to four random animals from the “privileged site” group and to four from the intradermal inoculation group. Five milliliters of serum was administered intraperitoneally to
FUTRELL,
ALBRIGHT,
ANI)
MYERS:
PREVEKTIOS
OF TUJlOIl
GROWTH
65
Fig. 8. Photograph of “alymphatic skill pedicles” of two strain 2 gllinra pigs 28 days after inocul:zt,ion with 1 X IO6 MCA-A tumor cells. The top animal, in addition, rcreived 1 X 109 immune sytrgeneic spleen cells intraperitoneally and was protected against tumor growth. The hottom animal received II~JITII:~~ syngeneic spleen cells and has a large, progressively gro”.ing “alymphatic pedicle tumclr.”
DAYS
AFTER
TUMOR
INOCULATION
Fig. 5. Growth of 1 X lo6 MCA-il tumor cells in alymphatic skin pedicles of syngeneic strain 2 guinea pigs after adoptive transfer of tumor-specific transplantation immilnity by intraperitoneal administration of immune spleen cells.
each animal on five occasions--% days prior to tumor inoculation and 1, 4, 7, and 11 days after inoculation. The remaining animals in each group received a similar volume and administration schedule of normal isologous serum. Large progressive tumors grew in each
of the animals receiving tumor inocula into t,he alyrnphat’ic “privileged sites,” regardless whether they received normal or immune serum (Fig. 5). In addit’ion, of the animals receiving the normally subgrowth intradermal tumor inoculat,ion and normal isologous serum
66
JOURNAL
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VOL.
lJpLb23 %%e 2 Spleen Cells
Strain Immune Node
Strain
2
Fig. 4. Diagrammatic scheme demonstrating failure of spleen or lymph node cells from syngeneic animals tumor growth when to prevent “privileged site” given intradermally or subcutaneously to nonimmune animals.
MCA-A Hyperlmmune
%
2,
FEBRUARY
1972
The concept of immunologic reactivity relative to a defense mechanism against cancer is not new. Thomas [15] init’ially suggested that from a phylogenetic viewpoint the survival of multicellular organisms was dependent upon a cont,rol system of cell-mediated immunity to recognize and reject. mutant. cells. By such a mechanism the genetic stability of the species could thereby be maintained. In his view, abrogat,ion of this function resulted in neoplasia while its efficient operation prot)ected against this hazard. Such a postulate has been elaborated and termed “immunological surveillance” by Burnet [3] to describe the immune eliminat’ion of microscopic foci of incipient tumors. Increasing clinical and experimental evidence suggests that such a concept has validity; (1) in the presence of clinical systemic immunosuppression t’he development and progression of spontaneous neoplasms is increased [13] (2) in I&O evidence suggesting the presence of tumor-specific cellular and humoral immunity associated wit’h certain human neoplasms [S, 11, 161. The crit’ical role of the lymphatic system relat’ive to “immune surveillance” in the skin allograft rejection process has been well
2 Lymph Cells
%
NO.
DISCUSSION
all failed to grow intradermal tumors (O/4). Two of the four animals receiving the immune serum, however, developed intradermal tumors in the int’act flank area.
2
12,
Normal
%
%
%
%
Fig. 6. Diagrammatic scheme demonstrating attempts to confer tumor-specific transplant,ation immunity by multiple inocula of spleen and lymph node cells and serum from syngeneic animals previously rejecting the MCA-A tumor. All animals receiving intradermal or subcutaneous immune cells or intraperitoneal immune serum grew large “privileged site” tumors.
FUTRELL,
ALBRIGHT,
AND
MYERS:
PREVENTION
OF TUMOR
67
GROWTH
Strain 2 MCA-A
5ml+ Serum I.f? x5 Days- 2,tl,t4,+7+11
5 ml + Serum 1.P x 5 Days -2,+1.+4,+7,+11 \ 0 MCA-A Hyperimmune
Tumor (50%)
CD Normal
No Growth
Fig. 6. Diagram showing enhanced tumor growth from a normally subgrowth 5 X lo5 MCA-A tumor cells in animals receiving serum from MCA-A-immune ceiving normal isologous serum failed to grow tumors.
demonstrated by Barker and Billingham [l]. Using the “alymphatic skin pedicle” model, a system first proposed by Frey and Wenk [5] in 1957 to study delayed hypersensitivity reactions, bhey were able to show convincingly that guinea pig skin allografts across a strong histocompatibility barrier were indefinitely prolonged so long as afferent lymphatic disruption was maintained between the graft and recipient animal. When lymphatic continuit,y was reestablished, as long as 100 days after grafting, the skin allograft nas promptly rejected. In addition, animals harboring a skin allograft’ on an “alymphatic pedicle” rejected a second orthot)opic skin graft from the same donor in first-set fashion suggesting that, they were not sensitized by the first allograft. Employing a similar experimental design, although using rats rather than guinea pigs, Tilney and Gowans [14] confirmed t’hat afferent lymphatic disruption conferred considerable skin allograft. prolongation although in their system eventual rejection and sensitization did occur. Using tumor allografts of a chemical carcinogen (20-methylcholanthrene)-induced t,umor we [6] have recently further demonst,rated that, the regional lymphatics play an essential role in the early detect,ion and rejection of allogeneic tumors. When the host animals “im-
intradermal tumor sytrgenric animals.
inoculum httimals
of re-
mune surveillance” mechanism is disrupted by interrupting the affercnt lymphatics from the inoculation site, the tumors attained an average volume of more than 500 times that, of identical tumor inocula transplanted to skin sites with normal lymphatic drainage. Unlike the skin allograft model reported by Barker and Billingham [I], however, the host does become sensitized to the tumor allograft even without the necessity of local lymph node participation. When syngeneic tumors (induced and maintained by serial transplants in straiIl-2 guinea pigs) are t,ransplanted to the “alymphatic skin pedicle” privileged site, a normally subgrowth tumor inoculum will grow progressively resulting in death of the animals [7]. Amput ation of t,he “privileged site tumors” after “0 days residence on an “alymphatic pedicle,” however, sensitized the a.nimals to the tumorspecific transplantation antigens of the syngeneic tumor and rendered them immune to further challenge with syngeneic t.umor transplants, even to a second privileged site. As on extension of this work, the present study was undertaken to determine the feasibility of preventing tumor growth in &II immunologically “privileged site” bg administrat#ion of cells or serum from isologous anima,ls
68
JOURNAL
OF
SURGICAL
RESEARCH,
previously rejecting a MCA-A tumor graft. By such a process we were able to demonstrate that 1 X log spleen cells from isologous MCA-A-immune animals (animals previously reject,ing at least six weekly intradermal challenges of 1 X IO6 NCA-A tumor cells) were able to adoptively transfer tumor-specific immunit.y and thereby prevent bumor growth in the surgically created “privileged sit,e.” It was not possible in our system, however, to prevent tumor occurrence in the alymphatic “privileged site” by passive transfer of tumor immunity using serum from immune animals. Indeed, when the immune serum was administered to animals receiving the same normally subgrowt’h intradermal tumor inoculum enhanced growth occurred. This finding supports the concept that cell-mediated immunity is primarily responsible for t,he immunologic rejection of chemically induced syngeneic tumor graft’s. The efficacy of transfer of cell-mediat,ed tumor-specific immunity to unimmunised animals was variable, however. The route of delivery of the immune cells to t’he test animals was critical in that those immune spleen cells given intraperitoneally were effective in preventing tumor growth while those given intradermally or subcutaneously in the same quantity did not prevent t)umor occurrence in the “privileged site.” The single tumor that did occur on the alymphatic skin pedicle after int,raperitoneal immune spleen cell administration was significantly smaller than those occurring on animals receiving eit’her isologous, nonimmune spleen cells or no spleen cells. These studies emphasize t,he importance of the afferent Iymphatics in the “immune surveillence” mechanism of tumor recognition and reject’ion. Whether neoplasia ever preferentially arises de novo in areas deprived of normal, functional afferent lymphatic drainage is conjectural. When syngeneic t,umors are transplanted to such immunologically “privileged sites,” however, passive transfer of tumorspecific immunit,y employing intraperitoneal administrat,ion of immune spleen cells can be effectjive in preventing tumor growth. SUMMARY The critical role of the afferent lymphatics in the early recognition and reject’ion of a meth-
VOL.
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NO.
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FEBRUARY
1972
ylcholanthrene-induced liposarcoma is demonst,rated. Prevention of tumor growth in an immunologically “privileged site” lacking normal afferent lymphatic drainage was possible by adoptfive transfer of cell-mediated tumorspecific immunity using intraperitoneally administered immune spleen cells. Neit’her immune spleen cells nor immune lymph node cells n-ere successful in conferring such immunity when administered intradermally or subcutaneously. Serum from animals previously rejecting the syngeneic tumor likewise was not successful in prot,ect.ing against “privileged site” tumor growt,h. REFEREKCES It. E. Role of the 1. Barker, C. F., and Billingham, afferent lymphatics in the rejection of skin homografts. J. Ezp. IVecl. 128:197, 1968. 2. Billingham, R. E., and Silvers, W. K. Studies on cheek pouch skin homografts in the Syrian hamster. Ciba Foundation Symposium on Transplantation. In G. E. W. Wolstenholme and M. P. Cameron (Eds.), Transplantation, p. 90, London: Churchill, 1962. sur3. Burnet, F. M. The concept. of immunological veillance. Progr. Erp. Tumor Res. 13 :I, 1970. D. L. Impaired im4. Eilber, F. R., and Morton, munologic reactivity and recurrence following cancer surgery. Cancer 25:362, 1970. studies 5. Frey, J. R., and Wenk, P. Experimental on the pathogenesis of contact eczema in the guinea pig. Znf. Arch. Allerg. 11:81, 1957. J. W., and Myers, Jr., G. H. Role of the G. Futrell, regional lymphatics in tumor allograft rejection. Transplantation (in press). 7. Futrell, J. W., and Myers, Jr., G. H. The regional Annals of lymphatics and cancer immunity. Sztrgeq (submitted for publication, 1971). IX. E., and Hellst.ldm, I. Cellular ima. Hellstriim, munity against tumor antigens. Advan. Cancer Res. 12:167, 19G9. D. L. 9. Holmes, E. C., Kahan, B. D., and Morton, Soluble tumor-specific transplantation antigens from methylcholanthrene-induced guinea pig sarcomas. Cancer 25:373, 1970. R. E., and Burke, D. Production of 10. Madden, viable single cell suspensions from solid tumors. J. Nat. Cancer Inst. 27:841, 1961. 11. hlorton, D. L., Eilber, F. R., Joseph, N. L., Wood, W. C., Trahan, E.. and Ketchan, A. S. Immunological factors in human sarcomas and melanomas: -4 rational basis for immunotherapy. Ann. Surg. 172740, 1970. 12. Raju, S., and Grogan, J. B. Immunology of anterior chamber of the eye. Transplant. 23-0~. 3 fiO5, 1971. 13. Southam, C. E. Evaluation of the immunologic
FUTRELL,
ALBRIGHT,
ANI> LITERS:
capability of cancer patients. Cancer Res. 28: 1455, 1968. 14. Tilney, N. L., and Goa-ans, J. L. The sensitization of rats by allografts transplanted to alymphat,ic pedicles of skin. ,J. Exp. Med. 133:951, Nil. 15. Thomas, L. Cellular and humoral aspects of the
PREVENTION
0~ TUMOR
GROWTH
69
hypersensitivity state. In H. S. Lan-renw (Ed.), “Cellular and Humoral Aspects of the Hyperscnsitivit.y St.atc” Discr~ssion, p. 529. iYcw York Harper (Hoeber), 1959. 16. Lanky, F., Stjernsward, and Nilsonne, W. Cellular immunity to human sarcoma. J. Snl. C’nncer Inst. 16:1145, 1971.
OF
SURGICAL
RESE.4RCI1,
12,
62-69
(1972)
PREVENTION OF TUMOR GROWTH IN AN “IMMUNOLOGICALLY PRIVILEGED SITE” BY ADOPTIVE TRANSFER OF TUMORSPECIFIC TRANSPLANTATION IMMUNITY J. WILLIAM
FUTRELL,
M.D.,
NILE
L.
ALBRIGHT,
GEORGE
MATERIALS
H.
MYERS,
JR.,
M.D.
AND
filETHODS
Inbred female Sewall-Wright strain 2 guinea pigs, obtained from the Laboratory Branch of the National Institutes of Health, were used. These animals uniformly accept first and second set isograft,s. In each experiment,, all animals were age-matched and housed in group cages containing six animals. Water and Wayne guinea pig chow with supplemental ad libif~~~~~. A methylcarrots were available cholanthrene-induced liposarcomn, dcsignat,ed MCA-A, and shown to possess tumor-specific transplantation antigens [9], n-as used in all experiments. The tumor was inducaed by subcubaneous injection of 4.0 mg. ?IICd and has been maintained in our laboratory by periodic transplantation int.0 female strain 2 guinea pigs. All experiments employed tumor grafts earlier than the tenth transplant generation. Using our own modification of a procedure devised by Frey and Wenk [.5], and later adapted by Barker and Billingham [l], circular, full-thickness skin pedicles approximately 4 cm. in diameter and containing the panniculus carnosus muscle were cut’ from the shaved flanks of ether-anesthet’ized guinea pigs. A prominent neurovascular stalk supplying t,he isolated segment of flank skin, and comprised of a perforating intercost,al artery and vein with some adjacent neural fibers, n-as identi-
From The Surgery Branch, National Cancer InstiNational Institutes of Health, Bethesda, tute, Maryland. Submitted for publication November 19, 1971. 62 Q 1972 by Academic Press, Inc.
AND
elucidated, however. To this end a model was developed to study the effect of adoptive t,ransfer of tumor-specific transplantation immunity on the growth of a syngeneic tumor in a surgically created immunologically “privileged site” in in-bred guinea pigs.
EVIDENCE CONTINUES TO ACCUAIIJLATE that t,he development of human ncoplastic disease is in considerable measure relatcd to the degree of immunocompetence of the host organism [4]. It is now well established that in the presence of systemic immunodepression, whether from drugs, irradiation, or other causes, the liklihood of development and progression of cancer is considerably incrcascd [13]. Based upon experimental work demonstrating increased tumor growth in areas totally deprived of afferent lymphatic drainage, we have proposed that it is conceivable that neoplasia may grow preferentially in areas of “localized immunosuppression” in which a normally immunocompetent host is unable to recognize the foreign antigenicit,y of tumor cells [6, 71. A unique experimental model for studying the capability of an organism to immunologically recognize and reject genetically nonident,ical t(issue grafts has been the use of transplants to so-called immunologically privileged sites [I, 2, 141. Such arcas lack normal, funct,ional afferent, lymphatic drainage and are, by definition, anatomic sites within the host milieu where foreign tissue is protected against the normal immunologic rejection process and allowed a prolonged survival. Perhaps the best studied examples of such sites are the hamster check pouch [2] and the ant.erior chamber of the eye [12]. The role of “immunologically privileged sites” relative to t)umorspecific transplantation immunity has not been
Copyright
M.D.,
FUTRELL,
ALBRIGHT,
AND MYERS:
fied and isolated. Any adherent areolar tissue or connecting fibers was sharply excised. The resulting circular skin defect was then closed longitudinally with interrupted 3-O nylon sutures leaving a small cent,ral aperture for emergence of the vascular pedicle. A double sterile sheet of Adaptic nonadherent dressing (Johnson and Johnson, New Brunswick, KJ) with a cent#ral slit to accommodate t,he vascular stalk was placed over the wound and under the raw surface of the skin flap. The pedicle was t,hen repositioned on the flank and stabilized with surrounding adhesive tape. An additional sterile dressing of Telfa (Kendall, Chicago, IL) was applied with multiple strips of wat’errepellent adhesive tape. The dressing allowed normal mobilit,y and prot’ection of the pedicle from cannibalism. The adequacy of complete aff erent lymphat,ic int.erruption in this model has previously been reported [I, 61. A single-cell suspension of the MCA-A tumor was prepared by t,rypsin enzymatic dissociation of the solid tumor according to the technique of Madden and Burke [lo]. This process yielded a suspension cont.aining 90-95 % viable tumor cells as judged by trypan blue (0.05 70) exclusion. The cells were suspended in media RPM1 with 20 % fetal calf serum and the conc&rat,ion adjusted so that 0.05 ml contained the desired number of viable tumor cells. A Zgaugc needle 011 a tuberculin syringe was used for all inject,ions. Meticulous dressing daily and t,hereafter less changes, initially frequently, \vcrc necessary to assure good viability of the pedicle with no chance for reatt.achment.. Thrice weekly inspection and palpation of the pedirles was employed to dctermine the time of initial appearance of tumor nodules. After this, measurement of the two largest perpendicular diameters of the tumor was performed. After each inspection, careful redressing was performed. Additional normal strain 2 animals lvere injected weekly for (i-10 weeks with a subgrowth intradermal inoculum of 1 X lo6 NCA-A tumor cells for preparation of immune cells and serum for adoptive t,ransfer of tumorspecific transplantat,ion immunity. The animals were exsanguinated by intracardiac blood aspiration and serum was obtained by centrifugation of the whole blood at 200g for 10 minutes.
PREVENTION
OF TUMOR
G3
GROWTH
After t,his spleen cell suspensions were made from spleens aseptically excised from t,hese MCA-A immune strain 2 animals. The spleens were individually minced with scissors in Earle’s balanced salt solution and passed through 40-mesh stainless-steel screens. The suspension was then filtered through SO-mesh stainless&eel screens and centrifuged at. 2OOg for 10 minutes. The cell button was resuspended in media RPM1 1640 with 20% fetal calf serum. Viability was determined by trypan blue exclusion and the concentration adjust,ed to contain 1 X log viable spleen cells. Lymph node cells were prepared in a similar manner from the axillary and inguinal nodes of multiple RICh-A-immune animals. EXPERIRIE?;TS
ASD
RESULTS
Experiment 1. Precentiojl of “Prizdegecl Site” Tumor Growth by ;ldoptive Tratisjer ctf CellWe have previously shown that 1 X lo6 MCA-A tumor cells will grow progressively in a syngeneic host resulting in death of the animal when inoculated into an al~mphntio skin pediclc “privileged site” [7]. The same size tumor inoculum is routinely rejected n-hen given intradermall\r in an area with intact lymphatic drainage. In order to assess the effectiveness of passive adminisr rution of immune spleen cells in protecting ngainst such t,umor growth in an immunologically privileged site, the following experiment was performed. Alymphatic skin pcdicles were constructed 011 the flanks of nine animals and randomlg inoculated with 1 X lo6 viable syngencic MCA-A tumor cells (I’ig. 1). In addition, three animals received an intraperitoneal injection of 1 X log immune spleen cells from isologous animals previously rejecting multiple intradermal inocula of MCA-A tumor cells. Tllree animals received 1 X log nor1na.l spleen cells intraperitoneally from isologous animals while the remaining animals received no spleen cells. Large progressive tumors grew on the alymphatic pedicles of all animals receiving either no spleen cells (3/3) or normal isologous spleen cells (3/3) (Fig. 2). Two of the three animals receiving intraperitoneal immune isologous spleen cells were protected against “privileged
64
JOURNAL
-No
growth
OF
Tumor
SURGICAL
RESEARCH,
Tumor
Fig. 1. Diagrammatic scheme of Exp. 1 showing inoculation of 1 X lo6 MCA-A syngeneic t,umor cells into alymphatic skin pedicles of strain 2 guinea pigs. Those animals which, in addition, received an intraperitoneal injection of 1 X lo8 spleen cells from syngeneic animals previously rejecting the MCA-A tumor xere protected against “privileged site” tumor growth. Animals which received normal syngeneic spleen cells or no spleen cells grew large tumors.
site” kunors. The one t’umor that occurred in an animal receiving intraperitoneal immune spleen cells was significantly smaller t’han those of the controls (Fig. 3). Experiment 2. Failure of Immune Spleen or Lymph Node Cells to Confer Tumor-SpeciJic Immunity When Administered Introdermally or Subcutaneously to Nonimmune Animals Having established that spleen cells from MCA-A immune animals were capable of adoptive transfer of tumor-specific transplantation immunity to an immunologically “privileged site”, the following experiment was designed to determine the effect of the route of administration of the immune cells on the de-
VOL.
12,
NO.
2,
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1972
gree of conferred immunity. Alymphatic skin pedicles were constructed on nine animals and inoculated with 1 X lo6 MCA-A tumor cells (Fig. 4). Three random animals also received an int,radermal/subcut’aneous inoculation of 1 X log immune isologous spleen cells, while I hree received 1 X log intradermal/subcut,aneous lymph node cells from MCA-A-immune isologous animals. The remaining animals received no immune cells. All animals (9/9) gre\\ large, progressive “privileged site” tumors. In an attempt to demonstrat,e some protcction against “privileged site” tumor growth by adoptive transfer of intradermally/subcutaneously administered immune spleen and lymph node cells, the above experiment leas repeated using one-half the number of tumor cells (5 X lo5 I\ICA-A) and multiple inoculations of the immune spleen and lymph node cells. The immune cells were harvested and administered on three separate occasionsonce 2 days prior to tumor inoculation and 1 and 4 days aft,er tumor inoculation. Again no tumor prevention was demonstrated, however, in that all animals grew large “privileged site” tumors (Fig. 5). Experiment S. The E$ect of Isologous LLImmune” Serum on MCA-il Tumor Growth Although cell-mediated immunity is thought to be the primary component in the immunologic rejection of chemically induced syngeneic tumor grafts, a series of animals was studied to define the effect of serum from animals previously rejecting the MCA-A tumor on syngeneic tumor growth in an immunologically “privileged site.” Alymphatic skin pedicles were constructed on eight animals and inoculated with 5 X lo5 MCA-A tumor cells. Eight additional animals received a normally subgrowth intradermal inoculum of 5 X IO5 MCA-A tumor cells in an area with intact lymphatics. Immune serum from isologous animals previously rejecting multiple intradermal MCA-A tumor inocula was administered to four random animals from the “privileged site” group and to four from the intradermal inoculation group. Five milliliters of serum was administered intraperitoneally to
FUTRELL,
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Fig. 8. Photograph of “alymphatic skill pedicles” of two strain 2 gllinra pigs 28 days after inocul:zt,ion with 1 X IO6 MCA-A tumor cells. The top animal, in addition, rcreived 1 X 109 immune sytrgeneic spleen cells intraperitoneally and was protected against tumor growth. The hottom animal received II~JITII:~~ syngeneic spleen cells and has a large, progressively gro”.ing “alymphatic pedicle tumclr.”
DAYS
AFTER
TUMOR
INOCULATION
Fig. 5. Growth of 1 X lo6 MCA-il tumor cells in alymphatic skin pedicles of syngeneic strain 2 guinea pigs after adoptive transfer of tumor-specific transplantation immilnity by intraperitoneal administration of immune spleen cells.
each animal on five occasions--% days prior to tumor inoculation and 1, 4, 7, and 11 days after inoculation. The remaining animals in each group received a similar volume and administration schedule of normal isologous serum. Large progressive tumors grew in each
of the animals receiving tumor inocula into t,he alyrnphat’ic “privileged sites,” regardless whether they received normal or immune serum (Fig. 5). In addit’ion, of the animals receiving the normally subgrowth intradermal tumor inoculat,ion and normal isologous serum
66
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Fig. 4. Diagrammatic scheme demonstrating failure of spleen or lymph node cells from syngeneic animals tumor growth when to prevent “privileged site” given intradermally or subcutaneously to nonimmune animals.
MCA-A Hyperlmmune
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The concept of immunologic reactivity relative to a defense mechanism against cancer is not new. Thomas [15] init’ially suggested that from a phylogenetic viewpoint the survival of multicellular organisms was dependent upon a cont,rol system of cell-mediated immunity to recognize and reject. mutant. cells. By such a mechanism the genetic stability of the species could thereby be maintained. In his view, abrogat,ion of this function resulted in neoplasia while its efficient operation prot)ected against this hazard. Such a postulate has been elaborated and termed “immunological surveillance” by Burnet [3] to describe the immune eliminat’ion of microscopic foci of incipient tumors. Increasing clinical and experimental evidence suggests that such a concept has validity; (1) in the presence of clinical systemic immunosuppression t’he development and progression of spontaneous neoplasms is increased [13] (2) in I&O evidence suggesting the presence of tumor-specific cellular and humoral immunity associated wit’h certain human neoplasms [S, 11, 161. The crit’ical role of the lymphatic system relat’ive to “immune surveillance” in the skin allograft rejection process has been well
2 Lymph Cells
%
NO.
DISCUSSION
all failed to grow intradermal tumors (O/4). Two of the four animals receiving the immune serum, however, developed intradermal tumors in the int’act flank area.
2
12,
Normal
%
%
%
%
Fig. 6. Diagrammatic scheme demonstrating attempts to confer tumor-specific transplant,ation immunity by multiple inocula of spleen and lymph node cells and serum from syngeneic animals previously rejecting the MCA-A tumor. All animals receiving intradermal or subcutaneous immune cells or intraperitoneal immune serum grew large “privileged site” tumors.
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GROWTH
Strain 2 MCA-A
5ml+ Serum I.f? x5 Days- 2,tl,t4,+7+11
5 ml + Serum 1.P x 5 Days -2,+1.+4,+7,+11 \ 0 MCA-A Hyperimmune
Tumor (50%)
CD Normal
No Growth
Fig. 6. Diagram showing enhanced tumor growth from a normally subgrowth 5 X lo5 MCA-A tumor cells in animals receiving serum from MCA-A-immune ceiving normal isologous serum failed to grow tumors.
demonstrated by Barker and Billingham [l]. Using the “alymphatic skin pedicle” model, a system first proposed by Frey and Wenk [5] in 1957 to study delayed hypersensitivity reactions, bhey were able to show convincingly that guinea pig skin allografts across a strong histocompatibility barrier were indefinitely prolonged so long as afferent lymphatic disruption was maintained between the graft and recipient animal. When lymphatic continuit,y was reestablished, as long as 100 days after grafting, the skin allograft nas promptly rejected. In addition, animals harboring a skin allograft’ on an “alymphatic pedicle” rejected a second orthot)opic skin graft from the same donor in first-set fashion suggesting that, they were not sensitized by the first allograft. Employing a similar experimental design, although using rats rather than guinea pigs, Tilney and Gowans [14] confirmed t’hat afferent lymphatic disruption conferred considerable skin allograft. prolongation although in their system eventual rejection and sensitization did occur. Using tumor allografts of a chemical carcinogen (20-methylcholanthrene)-induced t,umor we [6] have recently further demonst,rated that, the regional lymphatics play an essential role in the early detect,ion and rejection of allogeneic tumors. When the host animals “im-
intradermal tumor sytrgenric animals.
inoculum httimals
of re-
mune surveillance” mechanism is disrupted by interrupting the affercnt lymphatics from the inoculation site, the tumors attained an average volume of more than 500 times that, of identical tumor inocula transplanted to skin sites with normal lymphatic drainage. Unlike the skin allograft model reported by Barker and Billingham [I], however, the host does become sensitized to the tumor allograft even without the necessity of local lymph node participation. When syngeneic tumors (induced and maintained by serial transplants in straiIl-2 guinea pigs) are t,ransplanted to the “alymphatic skin pedicle” privileged site, a normally subgrowth tumor inoculum will grow progressively resulting in death of the animals [7]. Amput ation of t,he “privileged site tumors” after “0 days residence on an “alymphatic pedicle,” however, sensitized the a.nimals to the tumorspecific transplantation antigens of the syngeneic tumor and rendered them immune to further challenge with syngeneic t.umor transplants, even to a second privileged site. As on extension of this work, the present study was undertaken to determine the feasibility of preventing tumor growth in &II immunologically “privileged site” bg administrat#ion of cells or serum from isologous anima,ls
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previously rejecting a MCA-A tumor graft. By such a process we were able to demonstrate that 1 X log spleen cells from isologous MCA-A-immune animals (animals previously reject,ing at least six weekly intradermal challenges of 1 X IO6 NCA-A tumor cells) were able to adoptively transfer tumor-specific immunit.y and thereby prevent bumor growth in the surgically created “privileged sit,e.” It was not possible in our system, however, to prevent tumor occurrence in the alymphatic “privileged site” by passive transfer of tumor immunity using serum from immune animals. Indeed, when the immune serum was administered to animals receiving the same normally subgrowt’h intradermal tumor inoculum enhanced growth occurred. This finding supports the concept that cell-mediated immunity is primarily responsible for t,he immunologic rejection of chemically induced syngeneic tumor graft’s. The efficacy of transfer of cell-mediat,ed tumor-specific immunity to unimmunised animals was variable, however. The route of delivery of the immune cells to t’he test animals was critical in that those immune spleen cells given intraperitoneally were effective in preventing tumor growth while those given intradermally or subcutaneously in the same quantity did not prevent t)umor occurrence in the “privileged site.” The single tumor that did occur on the alymphatic skin pedicle after int,raperitoneal immune spleen cell administration was significantly smaller than those occurring on animals receiving eit’her isologous, nonimmune spleen cells or no spleen cells. These studies emphasize t,he importance of the afferent Iymphatics in the “immune surveillence” mechanism of tumor recognition and reject’ion. Whether neoplasia ever preferentially arises de novo in areas deprived of normal, functional afferent lymphatic drainage is conjectural. When syngeneic t,umors are transplanted to such immunologically “privileged sites,” however, passive transfer of tumorspecific immunit,y employing intraperitoneal administrat,ion of immune spleen cells can be effectjive in preventing tumor growth. SUMMARY The critical role of the afferent lymphatics in the early recognition and reject’ion of a meth-
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ylcholanthrene-induced liposarcoma is demonst,rated. Prevention of tumor growth in an immunologically “privileged site” lacking normal afferent lymphatic drainage was possible by adoptfive transfer of cell-mediated tumorspecific immunity using intraperitoneally administered immune spleen cells. Neit’her immune spleen cells nor immune lymph node cells n-ere successful in conferring such immunity when administered intradermally or subcutaneously. Serum from animals previously rejecting the syngeneic tumor likewise was not successful in prot,ect.ing against “privileged site” tumor growt,h. REFEREKCES It. E. Role of the 1. Barker, C. F., and Billingham, afferent lymphatics in the rejection of skin homografts. J. Ezp. IVecl. 128:197, 1968. 2. Billingham, R. E., and Silvers, W. K. Studies on cheek pouch skin homografts in the Syrian hamster. Ciba Foundation Symposium on Transplantation. In G. E. W. Wolstenholme and M. P. Cameron (Eds.), Transplantation, p. 90, London: Churchill, 1962. sur3. Burnet, F. M. The concept. of immunological veillance. Progr. Erp. Tumor Res. 13 :I, 1970. D. L. Impaired im4. Eilber, F. R., and Morton, munologic reactivity and recurrence following cancer surgery. Cancer 25:362, 1970. studies 5. Frey, J. R., and Wenk, P. Experimental on the pathogenesis of contact eczema in the guinea pig. Znf. Arch. Allerg. 11:81, 1957. J. W., and Myers, Jr., G. H. Role of the G. Futrell, regional lymphatics in tumor allograft rejection. Transplantation (in press). 7. Futrell, J. W., and Myers, Jr., G. H. The regional Annals of lymphatics and cancer immunity. Sztrgeq (submitted for publication, 1971). IX. E., and Hellst.ldm, I. Cellular ima. Hellstriim, munity against tumor antigens. Advan. Cancer Res. 12:167, 19G9. D. L. 9. Holmes, E. C., Kahan, B. D., and Morton, Soluble tumor-specific transplantation antigens from methylcholanthrene-induced guinea pig sarcomas. Cancer 25:373, 1970. R. E., and Burke, D. Production of 10. Madden, viable single cell suspensions from solid tumors. J. Nat. Cancer Inst. 27:841, 1961. 11. hlorton, D. L., Eilber, F. R., Joseph, N. L., Wood, W. C., Trahan, E.. and Ketchan, A. S. Immunological factors in human sarcomas and melanomas: -4 rational basis for immunotherapy. Ann. Surg. 172740, 1970. 12. Raju, S., and Grogan, J. B. Immunology of anterior chamber of the eye. Transplant. 23-0~. 3 fiO5, 1971. 13. Southam, C. E. Evaluation of the immunologic
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capability of cancer patients. Cancer Res. 28: 1455, 1968. 14. Tilney, N. L., and Goa-ans, J. L. The sensitization of rats by allografts transplanted to alymphat,ic pedicles of skin. ,J. Exp. Med. 133:951, Nil. 15. Thomas, L. Cellular and humoral aspects of the
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hypersensitivity state. In H. S. Lan-renw (Ed.), “Cellular and Humoral Aspects of the Hyperscnsitivit.y St.atc” Discr~ssion, p. 529. iYcw York Harper (Hoeber), 1959. 16. Lanky, F., Stjernsward, and Nilsonne, W. Cellular immunity to human sarcoma. J. Snl. C’nncer Inst. 16:1145, 1971.