Studies of Thymic Function with Emphasis on the Role of the Thymus in Oncogenesist

[CANCER RESEARCH 26 Part I, 551-574, April 1966]

LLOYDW. LAW

National Cancer Institute, Bethesda, Maryland

This presentation will be concerned with 2 general topics: organ to other sites occurs with a selective seeding in spleen (a) our present knowledge of thymic structure and function, lymph nodes, and other lymphoid organs. but particularly the latter, as revealed by the results of recent For a more detailed discussion of the ontogeny of the thymus experiments in several species of animals following early thymic and its microscopic anatomy, the reader is referred to the studies ablation, and (b) consideration of the precise role of the thymus in of Smith (97) and of Ruth et al. (92). the initiation and suppression of neoplastic growths. Pertaining to Thymic Structure and Function Origin and Early Structure of the Thymus In most species the thymus is located in the upper anterior The thymus is a compound organ consisting of 3 quite different part of the chest. Exceptions are the chicken and guinea pig. cell systems: (a) lymphoid cells, (b) reticulum cells, and (c) The absolute size varies from species to species but the absolute e[)ithehal cells. The latter 2 may be referred to as the epithelial size of thymic lobules appears to be remarkably uniform in the reticulum cell complex. The thymus in mammals arises as various species, suggesting that there may be a critical limit for paired structures from the endoderm of the 3rd and 4th branchial the size of a thymic lobule. The incompletely divided lobules are [)OUcheS. Thymic tissue on each side, along with parathyroid composed of a medulla of mixed cells of the ej)ithelial reticulum elements, migrates caudally and eventually reaches the upper complex and lymphoid cells surrounded by a cortex in which mediastinum. In some strains of mice deposits of thymic tissue tightly packed lymphoid cells are embedded in a meshwork of remain in the thyroid-parathyroid area as ectopic thymus and epithehal reticulum cells. The lymphoid cortex has certain probably become functional (49). The thymus remains irnrely features which set it apart from other lymphoid organs. The epithelial with a connective tissue capsule of mesenchyme until mitotic activity in lymphoid cells is higher by 5- to 10-fold than the end of the 2nd month in man and until sometime near 18â€”19 in lymphoid cells of spleen, lymph nodes, Peyer's l)atches, etc., days after fertilization in the mouse (3). and with advancing age this activity remains high. This high There has Leen a running controversy over the years con mitotic activity is probably determined by the unique epithelial cerning the origin of lymphoid elements in this embryonic reticulum cell elements of the thymus. The evidence for the role epithelial structure. Some authors maintained that thymic round of the medulla in cortical function has been reviewed by Metcalf cells were of mesodermal origin arising from mesenchyme outside (66). In the thymus cortex of normal animals, in contrast to the thymus with subsequent migration into the organ or from other lymphoid organs, lymphoid follicles and germinal centers mesenchymal cells which had migrated into the epithelial thymus. usually are not present and plasma cells are infrequent. Others contended that lymphoid elements arose directly from The rate of production of lymphocytes in the thymus appears reticulum epithelium. Recently Auerbach (3) analyzed the not to be influenced by antigenic stimulation, partial thymec morphogenetic events in thymic development in mouse thymus tomy, resection of other lymphoid organs or the presence of organ cultures at a time when epithelium and mesenchyme are multiple thymic grafts. Lymphoid cells within the thymus, how quite distinct and observed an in situ lymphopoiesis, apparently ever, are known to be subjected to the same hormonal regulation from epithelial components. Host migrating cells, thymus as lymphoid cells in other organs. Also the fact that there is no mesoderm, and generalized mesenchyme appeared not to con increase in mitotic frequency or regeneration of thymic frag tribute to this initial lymphoid population of cells. Timewise, this ments following partial thymectomy strongly suggests that differentiation precedes to appearance of lymphoid cells in other thymic lymphopoiesis is associated with an intrinsic stimulus lymphoid organs. It may therefore be considered that the thymus related to certain unique structures scattered throughout the is â€œthemajor primordium of the mammalian lymphoid systemâ€• thymus (see Metcalf, Ref. 66). and that migration of lymphoid cells from this primary lymphoid Certain other characteristic features of thymic lymphoid elements have recently been demonstrated by serologic methods. Thymic lymphocytes have been shown to possess distinctive 1 G. H. A. Clowes Memorial Lecture, presented at the 56th antigens (79, 87) and they have also been shown to have a dis Annual Meeting of the American Association for Cancer Research tinctive sensitivity to the cytotoxic effect of guinea pig serum on April 8, 1965, Philadelphia, Pennsylvania. (28) and of natural isoantibodies present in the sera of certain Received for publication December 1, 1965. normal mice (94).

APRIL 1966 551

Table 1 lists some of the structural and functional differences birth in this laboratory are shown in Table 2. Strain and age of between thymus and other lymphoid organs. mouse at thymectomy significantly affect the severity and onset Functionally, there is no doubt that lymphoid cells recovered of the â€œwastingâ€•syndrome. C3H mice commonly show diarrhea from the thymus are quite distinct from those of other sources and â€œruffledâ€•fur whereas these conditions have not been such as lymph nodes, spleen, and Peyer's patches (7) ; it is also observed among the other inbred strains used in this laboratory. clear that at least a major portion of thymic lymphoid cells are Some strains of mice do not show â€œwasting.â€•Signsof wasting or not special types of lymphoid cells, at least in the adult animal of disturbed growth patterns have not been observed in our and that thymic stem cells are being replaced continually by NIH (Swiss-Webster) strain following thymic removal, nor was immigrant cells (66). Lymphocyte migration studies show that there observed a peripheral lymphopenia (see Table 3) ; yet these there exists a slow but continuous afferent stream of cells to the neonatally thymectomized mice show a strikingly altered thymus from the circulation (24). The nature of these immigrant response to infection with LCM virus (57) and have a deficient cells and the site or origin is unsolved. Residence, however, with homograft rejection mechanism to neopla.stic growths (un in the microenvironment of the thymus appears to influence the published observation). An absence of wasting but a selective function of these cells (73). effect of neonatal thymectomy on certain immunologic responses has been observed also in C57BL/6J mice (9). Effects of Neonatal Thymectomy The striking â€œwastingâ€•describedin the hamster (Mesocricetus auralus) following neonatal thymectomy by Sherman et at. (95) The unique structural and functional characteristics of the thymus, its pattern of growth attaining maximum size very TABLE 1 early in life, the evidence that it represents the primordium of MORPHOLOGIC AND FUNCTIONAL DIFFERENCES lymphoid cell populations at least in the mouse, all suggest that BETWEEN THYMUS AND OTHER LYMPHOID the thymus indeed directs the immunologic development and ORGANS responsiveness of the mammal. That it is an organ of vital Th importance was shown by @Iiller(69) and by Martinez et at. (62) tissueLymphocytesSmallerEpithelial ymusNonthymic lymphoid who performed surgical removal of the thymus in laboratory mice within 24 hr after birth. The consequences of early thymectomy may be grouped under cellsPresentAbsentMitoticreticular 3 syndromes the relationship of one to the other of which is not activity5â€”lOXXHassall as yet completely understood : (a) failure of normal bodily corpusclesPresentAbsentLymphoidâ€˜S growth and early mortality (wasting) ; (b) lymphocyte depletion; andAbsentPresentgerminalfollicles (c) immunologic deficits. centersPlasma cellsInfrequentPresentSite The lethal â€œwastingâ€•syndrome has been observed in rats (40) of localantigenicâ€”+response and hamsters (95) as well as in certain strains of mice. The results obtained following thymectomy within 24 hr after

TABLE 2 MORTALITY IN NEONATALLY THYMECTOMIZED MICE OF DIFFERENT STRAINSC

of dead! age at death (days)RemarC3Hf/Lw2863/76StrainNo. littersNo. No. thymectomizedMean and range

incompleteC3Hf/Bi1015/22 (85%)58.4 (30â€”148)13

(68%)64.4 (40â€”97)2 incomplete 5 with ectopic thymic tissueBALB/c73/13

(23%)85 (80â€”95)3 incomplete 7/13 with ectopic thymic tissueC57BL/Ka611/21

(52%)66 (39â€”125)4 incomplete 3/3 with ectopic thymic tissueDBA/2929/35

incomplete(BL (83%)72.4 (44â€”84)6

incompleteNIH150/63Alivex C3H)F12047/54 (87%)94.0 (30â€”180)7

14 months

a Age at thymectomy: <24 hr.

552 CANCER RESEARCH VOL. 26

TABLE 3 EFFECT OF NEONATAL THYMECTOMY ON PERIPHERAL LEtJKOCYTES

of leukocytes/cu mmL/G StrainNo.Age (wk)No. ratioÂ°TotalLyznpho GranulocytesC3Hf/LwIntact

or sham214-67,320 (4,000â€”14,600)45402,7801.64Thymectomy at birth354â€”65,170 (2,000â€”10,500)13453,8250.35DBA/2Intactorsham8911,200

(7,750-12,900)60455,1551.17Thymectomy at birth20 (7,100â€”29,700) 0.43NIH 109 1119,620 15,990 479510,400 9,1950.89 (8,750-27,500)9220 (Swiss-Webster)Sham106â€”86,880

(5,300â€”10,600)29003,9800.75Thymectomy at birth106â€”86,430 (3,200â€”9,500)34702,9601.17

a L, lymphocytes; G, granulocytes. has not been observed in this laboratory nor by Roosa et at. (88). agent or its product. Neonatally thymectomized mice because of Immunologic deficiencies do occur nevertheless in these animals. their immunologic deficits are susceptible to laboratory in â€œWastingâ€•was usually not observed in any of the strains fections; germ-free mice did not â€œwaste.â€•Thisinterpretation of listed in Table 1 if thymectomy was performed at 3 days of age the results is reasonable. However both studies concentrated rather than at birth; yet, as will be described later, specific but principally upon the effects upon bodily growth; more should be striking deficits were observed. learned about the effect of thymectomy in germ-free animals It is easy to conclude that thymectomized animals of certain upon other well-established parameters. strains and under certain experimental conditions â€œwasteâ€• and die because they â€œbecomepreoccupied with massive bac Lymphoid Depletion terial or viral infectionsâ€• or because their immune responses are specifically impaired and they are therefore susceptible to Great variability was observed in our studies of the lymphoid intercurrent infection. A proportion of thymectomized mice of tissues of neonatally thymectomized C3Hf/Lw mice (49). some strains, particularly C3H, but not C57BL, show focal Although the spleens were not noticeably reduced in size except necrotic lesions of the liver and other organs caused by a hepato in those mice found dead spontaneously, a significant change was trophic virus (21, 49), but there is no evidence that wasting or the absence of, or great reduction in, the germinal centers within death results from this damage. As will be shown later immuno the white pulp. Instead of the normal centers of active lympho logic deficits in thymectomized mice are selective and by no cytopoiesis, pale, relatively noncellular areas of eosinophilic means total. Their responses to certain viral and bacterial reticulum cells were found. In the lymph nodes, germinal centers antigens are within normal range and they produce normal were rare or absent and pale areas composed of large faintly levels of y-globulin. eosinophilic cells were prominent. In many cases a plasma cell Sera of our C57BL/Ka mice which â€œwasteâ€•followingneonatal hyperplasia was noted in the medulla, while in others the medul thymectomy were compared with sera of NIH strain mice which lary sinuses were empty, and cells in the medullary cords were do not waste for the prevalence of certain murine viruses known reduced. Occasionally the usual architecture of the node was to infect colonies of mice (see Rowe et al., Ref. 90). There were obscured by large pale eosinophilic reticulum cells. The histology found no differences between these 2 groups, nor differences of the bone marrow usually appeared to be little altered although between thymectomized mice of both strains and their non erythrocytopoiesis was often inconspicuous in comparison with thymectomized litter mates in laboratory infections of pneu granulocytopoiesis. The most severe lymphoid depletion was monia virus of mice (PYM), Reovirus, Type 3, Theiler mouse found in severely â€œwastedâ€•C3Hmice. encephalomyelitis (GDV11), Sendai virus, mouse pneumonitis Necropsies performed on C3H mice at 2â€”3weeks following (K virus), or mouse hepatitis virus (MHV). thymectomy revealed development of lymphoid tissue, although Two groups of investigators (64, 105) have concluded from this was noticeably reduced in some animals in comparison with their studies of early thymectomy in germ-free or low-pathogen controls. It is clear that development of lymphoid tissues does in mice that the primary factor in the pathogenesis of the â€œwastingâ€• fact occur but is followed by a rather rapid depletion of lympho syndrome appears to be environmental, presumably an infectious cytes and lymphocyte precursors with near extinction in severely

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TABLE 4 PRINCIPAL IMMUNE RESPONSES INFLUENCED BY THYMECTOMY IN MAMMALS

Depressed Unaffected

I. Antibody formation Hemolysins (19S) and ag Hemocyanin (7S) glutinins (7S) to RBC Ferritin Typhoid H and 0 SSSa III (7S) Killed influenza Polyoma virus Bovine serum albumin (BSA) LCM@virus Human -y-globulin Phage T 4

II. Cellular sensitivity Skin reactions to tuberculin Allogeneic skiii grafts reactions Skin reactions to BSA Xenogeneic skin grafts Auto-allergic encephalomyelitis Adj uvant arthritis Meningeal lesions of LCM infection Graft-vs-host response Allogeneic skin and tumor grafts

@ a @55 pneumococcus polysaccharide. @ LCM, lymphocytic choriomeningitis.

wasted mice. In the neonatally thymectomized rat the histologic ANTIBODY RESPONSE IN NEONATALLY changes in lvmphoid organs were somewhat different (103). THYMECTOMIZED C3H MICE Most strains of thvmectomized mice characteristically show a HEMOLYSIN persistently low peripheral blood-lymphocyte level. This is AGGLUTININ

attained at different times after thvmectomy depending upon 640 00000 0 strain. In some strains (see Table 3) the lvmphopenia is accom panied by granulocytosis. In others, for example, NIH (Table 320 00000 3), lymphoid depletion does not occur; yet neonatally thymec 000 160 @ tomized NIH mice are protected against the lethal effect of 000000 00000 â€¢â€¢ LCM virus (57) and show a strikingly deficient response to 80 - 00000 000 â€¢ allogeneic tumor grafting (unpublished observation). The signifi LU I-. 40 - 88888 â€¢.. 000 cance of the morphologic changes in relation to immunologic I-. reactivity therefore remains to be determined. U- 20 .... 00 â€¢S 0 Immunologic Deficits -J I0 C) @\Iuch of the early literature im[)hed that all of the immune 0 0 - .%%%%@% 0000 11= responses of neonatally thymectomized animals were impaired; a. C, HEMOCYANINFERRITIN.0-..0 it is clear now, however, that whether or not a response is LU obtained del)ends upon the strain of animal used, the test system 280 employed, and Ul)Ofl the nature and extent of the antigenic stimulus. 320 The immune deficiencies characteristic of several animal .-.0â€¢..-0..0000â€¢â€¢000â€¢â€¢ species resulting from thymic removal at birth are those con 80 cerned with (a) antibody formation and (b) cellular sensitivity 20 responses, including homograft rejection. 0â€¢000.-..- Some of the principal immune responses influenced by thymec 0 tomy at birth in the mouse and rat are shown in Table 4. Immune C T CT responses are not uniformly impaired. Although it has been observed in this laboratory that C3H mice provide a poor or CHART 1. See Fahey et al. (23) for details. absent antibody response (hemolysins and agglutinins) to sheep erythrocytes (53), these mice are capable of responding in nearly are compatible, according to Humphrey (37), with the hypoth normal fashion to ferritin and hemocvanin (23) (see Chart 1) and esis that the thymus in early life acts as a source of potentially to T2 phage clearance. Humphrey et al. (38) have reported that immunologically competent cells and that some random â€œseed thymectomized mice can produce normal antibody titers to ingâ€•of other lymphoid tissues had occurred before thymectomy; hemocyanin and pneumococcus Type III capsular polysaccharide, â€œseededâ€•cells had a restricted range of immunologic corn whereas loor immune responses were observed to other antigens, petence. including Salmonella typhi â€œHâ€•andâ€œ0â€•antigens.These findings The ability to reject allogeneic skin grafts and allogeneic

554 CANCER RESEARCH VOL. 2&

Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 1966 American Association for Cancer Research. Thymic Function tumor cells appears to be rather uniformly impaired as a result TABLE 5 of neonatal thymectomy in the mouse. It has been reported that IMMVNOGLOBULIN LEVELS IN NEONATALLY some inbred strains fail to reject allogeneic skin grafts across the THYMEcTOMIZED C3H MICEa strong 11-2 histocompatibility barrier or even xenogeneic (rat) skin grafts : yet it has been observed in this laboratory that (17 LITTERMATES) (17) (mg/mI) C57BL/Ka mice thymectomized at birth and exhibiting extreme COMPONENTCONTROL (mg/ml) â€œwastingâ€• and lymphoid depletion are still quite capable of Range7S Mean RangeTHYMECTO3&IZEDMean rejecting skin grafts of closely related H@2b donors such as Strain 129 and C57L. Apparently therefore some strains of mice -y2-globulin and particularly C57BL mice mobilize their limited resources 7S-yi-globulin 3.3 (1.3â€”5.9) 1.3 (0â€”4.2) extremely well. -YIA(ISIA)-globulin 1.7 (0.7â€”3.7) 1.8 (0â€”5.8) The immunologic capacity has been studied of lymphoid cells, @ylM-globulin1.3 1.1(0.7â€”2.2)(0.5â€”2.1)0.6 1.4(0.1â€”1.4)(0.4â€”3.3) principally of splenic origin, obtained from neonatally thymec tomized mice (13). These experiments show that the immuno a Serum was obtained 7 days following injection of 0.1 ml 10% logic deficits resulting from thymectomy reside within the sheep RBC. See Fahey et at. (23) for details. lym@)hoid cells themselves and do not result from nons[)ecific effects produced within the environment of the animal. Graft TABLE 6 versus-host reactions as measured by â€œruntâ€•diseaseor by the TURNOVER (SYNTHETIC) HATEs OF 7S -y-GL0BULIN5 spleen assay method of Simonsen reveal a striking deficiency of IN THYMECTOMIZED AND CONTROL C3H MICE such cells. Furthermore, depending upon the strain of mouse i@:Intact AT mg/day/mouse used, it has been shown that thymectomy as late as 35 days â€œI-LABELED SAMPLE FROM:RATES interferes with the capacity to produce normal graft-versus-host miceIntact mice Thymectomized reactions. The question of the role of the thymus in acquired immune mice (9) 5.1 (9) tolerance is not as yet resolved. A study of the capacity of Thymectomized mice6.3 8.7 (4) 7.5 (9) lymphoid cells from neonatally thymectomized mice to induce tolerance has not been pursued adequately. There is some evidence however to suggest that the breakdown of tolerance 2. Syntheses of 7S.@1-and 7S@2-globulins were within normal occurs by means of a thymus-dependent mechanism. It has been rates; in those thymectomized mice in which low levels were recorded that thymectomy in adult mice tolerant to a specific encountered, an accelerated catabolic rate was responsible. antigen Prevented the reappearance of reactivity to that antigen 3. Serum @y1A(I32A)-glObulinlevels were increased but irregu (12). larly, while serum @y1M-globulinswere at normal levels. It is known that mice thymectomized as adults have no obvious 4. Immunoglobulins were similar to those of normal mice on defects in immune responses although there may occur diminished the basis of isoantigens, antigenic determinants detected with numbers of lymphocytes in the peripheral blood (65). When heterologous antisera, and skin-sensitizing ability. thymectomy at 2 months of age is followed by a lethal dose of The levels of the 4 major immunoglobulin groups were deter irradiation but protection is afforded by syngeneic bone marrow, mined quantitatively at periods between 4 and 7 weeks in these mice show immunologic defects as revealed by response to thymectomized C3H mice using specific antisera in an agar-gel allogeneic skin grafting and to sheep erythrocytes (73) ; non diffusion technic. The 71M-globuhns were normal. @y1A-Globulins thymectomized mice subjected to the same stress are quite were never decreased and occasionally were high, but the striking capable of responding in near-normal fashion. These results show increases recorded in C57BL mice (38) were not encountered. clearly that in the adult animal the thymus is required for The levels of 7572- and 7S.@i-globulins were variable but often reestablishing immune mechanisms in those situations resulting present in normal amounts. Where â€œwastingâ€•was severe, from massive lymphoid depletion from stress such as lethal including diarrhea, these globulins were decreased sometimes to ionizing radiation. 40% of the mean control values (Table 5). When synthetic rates for these immunoglobulins were determined, however, it was Immunoglobulin Studies in Thymectomized Mice found that these were within normal range (Table 6) and that the The nature of the immune defect(s) following neonatal thymec low levels were a reflection of an increased rate of catabolism or tomy in mice, rats, and hamsters is not understood. The altera loss from the body. tions, however, are now known to be only partial and perhaps The immunoglobulins of the neonatally thymectomized C3H selective. It was hoped that a study of possible qualitative or mice were found to be qualitatively normal. The Iga 1 and Iga 2 quantitative changes in immunoglobulins would more easily isoantigenic determinants (19), which reflect genetic differences help define the immune deficits. C3Hf/Lw strain mice, thymec between strains, were present in the immunoglobulin molecules of tomized within 24 hr of birth, and their intact littermates were thymectomized mice. Ouchterlony analyses using rabbit anti used throughout this study. The findings (23) which confirm and serums were used to compare the antigenic determinants present extend those of Humphrey et al. (38) may be summarized as on the separate classes of immunoglobulins, and no antigenic follows: differences were detected. Skin sensitization in the guinea pig is 1. The 4 classes of immunoglobulins, 7S@2-,7S.@-, @y1A($@)-, known to be a specific property of the 7S.,2-globulins. A purified and â€˜ylM-globuhnswere present. preparation isolated from the sera of neonatally thymectomized

APRIL 1966 555

mice was found to sensitize in the same manner as globulins from IIJI normal intact littermates. These findings indicate therefore that the immune defects 0 /I7/cc/(cirSHAM) 20 common to many rodents following neonatal thymectomy are Â£ j//@p@@//@Ã§ /4 not related to the mature function of the cells of the immune . B@e m@'row /8 @ system but must be related to maturation (differentiation) of the 20@ A Ã„Ã¡//m1,,,@ /8 a A@@c/c//*ir@Jsce//sII responsible cells. a Thj'n@c/om,@edes/y15

Restoration Procedures in Thymectomized Mice U)

A number of methods may be used to protect mice against the 15 - effects of thymectomy at birth. Presumably most of these z methods are effective in other species. The consequences of the U) different methods, using replacement of tissues, or cells, are w instructive, as will be seen in any attempt to delineate thymic 10 - function. 1. Syngeneic thymic grafts, either neonatal or adult will Lu prevent or reverse the effects of thymectomy. In C3H mice, for example, grafts given at or prior to 3 or 4 weeks prevent wasting, and such restored mice behave as immunologically normal mice 5- in rejecting homografts and responding to bacterial or protein antigens; grafting later than this period is ineffective (see Chart 2). In this laboratory allogeneic thymic grafts (C57BL to C3H) have not been completely successfuly in preventing the effects of thymectomy unless grafted within a week of thymic removal; j@ I I i I â€¢ I @0 2 4 6 8 however, it has been reported that allogeneic thymic grafts are AGEIN WEEKS effective in lethally irradiated adult mice supported by bone marrow therapy (72). The consequences of thymectomy in the CHART 3. Reconstitution, as measured by body weights in C3Hf/Lw mice thymectomized at birth. Dissociated cells from irradiated adult are somewhat modified, however, from the the several sources were administered i.v. 24 hr after thymectomy. consequences of thymectomy in the neonate. The striking effects Numbers after methods of treatment = number of mice in each of xenogeneic thymic grafting will be discussed later. group. See Trainin et al. (100) for details. IIIIIIIIIII 2. Dissociated lymphoid cells from adult syngeneic donors are BODY WEIGHT 25 effective in reconstituting neonatally thymectomized mice, but cells from different sources have varied potentialities. Such cells 20 have benefited from the presence of a thymus in the donor IS animal from which they were derived. As few as 10 < 106 cells from lymph node or spleen injected i.v. are sufficient to ensure .-6 U) IC normal growth and immunologic development, at least for a 4 QRAFT relatively long period. It is clear that newborn or adult lymphoid C, cells of thymic origin are less effective in reconstituting thymec z tomized mice, but under the proper conditions of time and dosage U) I- these too are known to be partially effective (83, 106). Several I . - . NORMAL CONTROLS C, parameters must be used to evaluate the patterns of reconstitu Lu ) A THYMECTOM/ZED tion (see Ref. 100). Chart 3 and Table 7 show some results of the 20 z effects of syngeneic adult lymphoid cells from various sources on 4 reconstitution of C3Hf/Lw mice thymectomized within 24 hr of Lu IS birth; lymphoid cells were injected i.v. at 48 hr. Of considerable I0 interest is the observation that many of these restored mice that @@@@@ I @::@@:_._â€˜@l.@â€˜10II nevertheless show near-normal growth patterns and immunologic 5 responsiveness died at 3â€”14 months following an acute â€œcachectic syndrome.â€• This suggests the likely possibility of depletion of the injected â€œstemâ€•cellsin these mice deprived of thymic tissue TIME IN WEEKS (100). 3. The protection afforded by syngeneic thymic grafts is most CHART 2. Influence on body weight of syngeneic neonatal thymic grafts in C3Hf/Lw strain mice thymectomized at birth. readily explained by assuming that the graft produces lymphoid Entire thymuses grafted into subcutaneous connective tissues at cells which â€œseedoutâ€• and consequently populate the host 3â€”4weeksor at 6â€”7weeks,as indicated by arrows. At least 10 mice lymphoid tissues and are in fact the cells directly responsible for for each point. recovery. Through the use of a cytogenetic marker, differ

556 CANCER RESEARCH VOL. 26

TABLE 7 RECONSTITUTION OF NEONATALLY THYMECTOMIZED MICE WITH DISSOCIATED LYMPHOPOIETIC AND HEMATOPOIETIC CELLSâ€•

GroupTreatmentmiceA of recipientsNo. and type of cells injectedNo. and % survivors at 8 wkDeath@:fingi@id@aI

thymectomy 0/35 (0%) B Neonatal thymectomy 18â€”20X106adult lymph node cells 13/13 (100%)) C Neonatal thymectomy 20â€”25X 10@adult thymic cells 36/37 (97%) D Neonatal thymectomy 10â€”30X 106adult bone marrow cells 18/22 (82%) at 7.4 (3â€”14)mo E Neonatal thymectomy 10â€”25X 106 adult spleen cells 35/35 (100%) F Neonatal thymectomy 15 X 10Â°neonatal thymic cells 8/14 (57%) @ GNeonatalNoneNone None 43/43 (100%)42/58 3/43 at 7,12,l3mo

a The strain used was C3Hf/Lw.

entiating host from donor tissue (71), or by employing the Several groups of investigators have now published results of discriminating spleen assay technic (13) in allogeneic thymic their studies and these are summarized in Table 8. In the early grafting, it was found that the majority of cells multiplying in studies (53, 56, 82) syngeneic neonatal or embryonic thymic host lymphoid tissues and after a period also in grafted thymus tissues in cell-tight but fluid-permeable Millipore diffusion were of host origin and could not have been provided by donor chambers of 0.3â€”0.45 @Laverage pore size were implanted i.p. into tissue. It should be pointed out, however, that these findings do neonatally thymectomized mice of several inbred strains. Pre not rule out the possibility that the thymic graft, particularly vention of â€œwastingâ€•andof lymphoid depletion and restoration within certain strains of mice, may indeed restore immunologic of immunologic competence were achieved in many of the recip function, at least in part, by itself providing potentially corn ient mice. The restorative effects of syngeneic thymic tissue in petent lymphoid cells (see Ref. 54). There must remain, therefore, Millipore chambers (0.45 @i) in C3Hf/Lw thymectomized within the thymectomized neonate a residuum of lymphoid cells recipients are shown on body weight (Chart 4), peripheral capable of becoming immunologically competent in the presence lymphocytes (Chart 5), lymphoid organs (Figs. 1, 2), and of the grafted thymic tissue. The proper maturation of lymphoid hemolysin production to sheep erythrocytes (Chart 6). cells under thymic influence could occur at distant sites through Later studies have included other strains of mice, lethally release of a humoral factor, or differentiation could occur within irradiated adults, and the use of other parameters for a study of the environment of the thymus by circulation of cells through restoration (Table 8). In addition, restorative effects of thymic the thymus. Subsequent colonization of these cells to lymphoid tissue enclosed within a Millipore chamber, have been recorded organs would follow. in the thymectomized rat (1). The pertinent observations to date may be summarized as Humoral Thymic Factor follows: Experiments were designed to determine whether a humoral 1. The data suggest the existence of a specific diffusible factor in thymic tissue exists and, if so, the nature of its influence. product from thymic tissue which enables the animal to produce

TABLE 8 RESTORATION IN THYMECTOMIZED MICEâ€”THYMIC TISSUE IN MILLIPORE DIFFUSION CHAMBERS

studiedReferencesC311,StrainMembrane pore size ration responses (IL)Resto (%)Immune

DBA0.4532Hemolysins to sheep RBCLevey et at. (56) (53)Swiss-Webster0.4551Response Law et at. (57)C57BL0.4590Response to LCMâ€•Levey et at. (52)CBA, to polyoma & Ting oncogenesisLaw 82)(BALB/c(CBA x T6)F,0.373Homograft rejection; & Miller (81, agglutinins to sheep rbcOsoba (5)C57BLb0.370Homograftx C57BL)F1Â°0.01â€”3.065Homograft rejectionBarclay ci at. (73)CBAC0. rejectionMiller et al. 1Homograft rejection ; (80) agglutinins to sheep RBCOsoba

a LCM, lymphocytic choriomeningitis.

b Thymectomy in adults plus total body X-radiation.

C Millipore chamber containing syngeneic lymph nodes or splenic tissue did not restore immune reactivity.

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TOTAL BODY WEIGHT PRIMARY HEMOLYSIN RESPONSE IN C3H MICE IIIIIIIII @NORMALCONTROLS 640 - â€¢.... S

U) S THYMECTOMIZED CONTROLS 0 THYMECTOMYAND DIFFUSION CHAMBER 55.. 55 4 320 â€” .ss... S 5555 @ 28 WITH THYMUSâ€”(ACTIVE) z 24 S [email protected] I-' I60 â€” Sâ€¢5@@â€¢ S 55555 @ 20 LU I- I- 80 â€” 5...5 S LA. z 12 0 4 w -j 4 55 U 40 â€” SSSSSssâ€¢ 5.5 â€” 0 4 0. S IIIIIIIII U LU 20- 5. 55555 5... 0 I 2 3 4 5 6 7 8 9 10 TIME IN WEEKS CHART 4. Body weight curves for C3H normal controls, thy 10 - mectomized at birth, and thymectomized mice bearing a diffusion chamber containing syiigeneic neonatal thymic tissue. Each point represents10mice. 0 â€” 55 .55555.55.. ss..ssâ€¢s.55555555 â€” IA AA TOTAL NUMBER OF LYMPHOCYTES INTACT THYMECTOMY THYMECTOMY THYMECTOMY IN THE PERIPHERAL BLOOD +1 SOLOGOUS + CHAMBER IIIIIIII THYMUS WITH THYMUS @ NORMAL CONTROLS CHART 6. Comparative hemolysin production determined at Day 7 among littermates of C3Hf/Lw mice. Isologous (syngeneic) 8000 â€” . ThYMECTOMIZEDCONTROLS neonatal thymic grafts were made into the subcutaneous connec 0 THYMECTOMY AND DIFFUSION CHAMBER tive tissues of the right axillary region. Millipore diffusion cham 7000 â€” WITh THYMUS hers containing syngeneic thymic tissue were transplanted i.p. at the same time as thymic grafting, that is 3-4 weeks. Only com 6000 pletely thymectomized mice (at birth) as checked at necropsy are @ 5000 included. See Law et at. (53) for details. 4000 z 3. Lymphopoiesis was not observed in thymic tissue within the chamber, and presumably only epithelial reticular tissue remained j 3000 0 4 (56, 82). This indicated that a humoral factor may be elaborated 2000 either by epithelial cells or possibly by specialized PAS2-positive I- reticular cells (67). @ 1000 4. In many restored animals only a small amount of tissue LU @ I' -IIIIIIII - was recoverable and was often found to be mostly necrotic at I 2 3 4 5 6 7 8 9 10 50â€”70days. This suggests that the influence of thymic tissue was TIME IN WEEKS required for only a limited, though critical, period. CHART 5. Absolute number of lymphocytes in the peripheral There is some evidence to suggest that THF (the thymic blood of 3 groups of mice, normal controls, thymectomized con humoral factor) is quite effective in inducing differentiation of trols, thymectomy with Millipore chamber containing syngeneic lymphoid precursor cells to immunologically competent cells neonatal thymic tissue. without stimulating lymphopoiesis. Restoration of immune reactivity has been observed in this laboratory (53), by Osoba new lymphocytes and to develop immunologic competence. (80), and by Isakovic et at. (39) in the absence of stimulated Immunologic capacity involving several responses was studied: lymphopoiesis. It may be postulated that THF induced immuno antibody production to protein antigens, homograft rejection of normal and neoplastic cells, delayed hypersensitivity, and graft 2 The following abbreviations are used: PAS, periodic acid versus-host reactions. Schiff; THF, thymic humoral factor; IP, immunologic potential; 2. The restorative effects were not attributable to a non IM, competent lymphoid cells; RSV, Rous sarcoma virus; CF, specific or adjuvant effect of the chamber (53, 57). Furthermore complement fixation ; MCA, methylcholanthrene ; MLV, Moloney tissue and lymph nodes were found ineffective in restoration (80). leukemogenic virus.

558 CANCER RESEARCH VOL. 26

TABLE 9 connective tissues. Restoration was achieved in a significant INFLUENCE OF XENOGENEIC THYMIC GRAFTS IN number of these F1 mice which are subject to â€œwasting,â€•but the (BL x C3H)F1 NEONATALLYTHYMECTOMIZED onset is relatively late and the condition relatively chronic. A MICE summary of the results using a group of parameters of restoration is given in Table 9. Growth curves are shown separately in Sham Chart 7. For details see Law (Restoration of Thymic Function in Neonatally Thymectomized Mice Bearing Xenogenic Thymic gm2.Mortality26.91.Body weight (15 wk) in 26.3Thymecto19.0Restored Grafts, to be Published) . Thymic grafts were not recoverable 20098%<10%54%days)3.Absolute(% survival at from any of the (Bi x C3H)F1 mice dying between 3 and 7 months or from those sacrificed at 7 months. The fate of these xenogeneic (at8715380062508no. lymphocytes grafts was obtained, however, in a special group in which recovery wk)4.Rejection was undertaken at 5-day intervals. Persistence of epithelial grafts100%26%70%byof AKR skin reticular cells was found in many grafts to as late as 40 days. 30days5.Mean to6.041.444.70sheeptiters of hemolysin Reconstitution with lymphoid cells into a typical thymic struc mo)(reciprocalsRBC (3 ture did not occur and there resulted eventually necrosis, invasion of log 2) by fibrous tissue, and rejection. This pattern is similar to that reported by Miller (72) and by Leuchars et at. (63) in their restoration studies using allogeneic thymic grafts introduced into thymectomized mice and again signifies the importance of persisting epithelial reticular elements of thymic tissue through a limited but critical period. 27 -

UI Discussion and Summary E 0 Chart 8 attempts to present a concept of possible origins of 0' 21 - immunologically competent cells in terms of thymic function. I- ,Th@mectomized in the mammal the major lymphoid l)rimOrdium is the thymus. I .__.o-â€”-â€”-â€” - -0 C-, Lymphocytes arise within the thymus either from mesenchymal . â€”U -I Lu elements or more likely from the epithehal complex as suggested â€˜Vhym.-notrestored 15 â€” CONCEPT OF ORIGINS OF IMMUNOLOGICALLY I I I I COMPETENTCELLS â€˜P @O 5 10 15 20 25 TIME (weeks)

CHART 7. Body weights in (Bl x C3H)F1 mice of the several groups : thymectomized at birth, sham-operated littermate con trols, thymectomized but restored following xenogeneic thymic graft, thymectomized but not restored following xenogeneic thymic grafting. Each point represents 15 mice. logic competence in those lymphoid precursors which left the thymus l)riOr to thymectomy. Our earlier work (56) and that to be discussed later concerning the effects of xenogeneic thymic grafts, nevertheless, suggests that THF under certain conditions also has a lymphocytosis-stimulating effect. That THF is not strain specific is shown by the work of Osoba (80). Allogeneic (C57BL) thymic tissue, enclosed within diffusion chambers, had the capacity to restore immune reactivity to skin homografting in CBA mice. This finding is in agreement with the earlier observations in thymectomized, lethally irradiated adult mice protected with syngeneic bone marrow (72). Allogeneic thymic grafts restored the capacity to produce homograft re actions, also rejecting thymus donor type skin as a 2nd set response.

THF also is apparently not species specific. (C57BL/KaLw x CHART 8. See text for discussion of suggested mechaiiisms con C3Hf/Lw)F1 mice were thymectomized at less than 18 hr after cerned with thymic function. Thymic factor = THF, thymic birth. Within 24 hr of thymectomy a single lobe of thymus from a humoral factor. IP, Lymphoid cells which have the potentiality newborn Osborn-Mendel rat was grafted to the subcutaneous to become competent ; IM, immunologically competent cells.

APRIL 1966 559

Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 1966 American Association for Cancer Research. Lloyd W. Law by Auerbach (3). The production of lymphoid precursors is most and donor are slight. Usually, depending upon the strain, intense during early life but must continued throughout life. lymphoid tissues are underdeveloped, and there is a concomitant There is some evidence that the thymic humoral factor (THF) deficiency of small lymphocytes throughout the body of neona stimulates and regulates lymphopoiesis and that this function tally thymectomized mice. is intrinsic to thymic tissue. Lymphoid precursors have immuno The animal with a deficient immune mechanism is useful for logic potential (IP) and some of these differentiate within the studies of host resistance to spontaneous and experimentally thymus and under the influence of THF into competent lymphoid induced neoplasms, particularly to those neoplasms reported to cells (IM) ; this is suggested by the results of reconstitution be antigenic in the syngeneic or autochthonous host and which experiments. The majority of cells, however, are IP and these might be expected to initiate a homograft-type reaction. The enter the circulation by way of the blood and lymph. THF also frequency and progression of neoplasms induced by certain acts upOfl distant lymphoid tissues, as shown by the diffusion chemical carcinogens and by polyoma virus are indeed reported chamber results, rendering potentially reacting cells (IP) com to be influenced by thymectomy (16, 17, 46, 74, 75, 77, 108). petent (IM). The existence in lymphoid organs of cells with Polyoma virus upon inoculation into newborn mice multiplies immunologic potential but incapable of responding immuno to high titer in many organs, produces an antiviral antibody logically is suggested from X-irradiation experiments of adult response, and after a fairly short latent period induces neoplastic thymectomized mice. Under the influence of THF in adults growths particularly in the salivary glands but in other tissues competence is induced in these cells. Clearly, therefore, recovery as well. C57BL and A strain mice, however, are resistant to the or reestablishment of lymphopoietic and immune functions in oncogenic effects of the virus introduced at birth and behave as the adult, particularly in times of stress, is dependent upon a adult animals of the susceptible strains despite the fact that thymus-controlled mechanism. THF appears also to be necessary polyoma virus again multiplies and antiviral antibodies are in the maintenance throughout life of normal immune functions. produced. That resistance to polyoma virus oncogenesis is related This is evident from the results of reconstitution experiments. to defense mechanisms operating at the level of the organism is Although adult syngeneic lymphoid â€œstemcellsâ€•restore neona shown by the ability of virus to transform organ explants of tally thymectomized mice to near normalcy, mortality is high in C57BL and A strain salivary tissue to neoplastic growths (C. J. later life among these â€œrestoredâ€•mice,signifying a finite life-span Dawe, unpublished observation). of the donated lymphoid cells in the absence of thymic tissue. The present discussion concerns results of studies designed to Since some of the immune responses of neonatally thymec gain some knowledge of the relation of immunologic competence tomized mice are not impaired, it is suggested that there exists to the induction of neoplasms by several methods but particu an additional site(s) for the origin of immunologically competent larly by polyoma virus. cells (nonthymic IM). These cells mature and differentiate with out intervention of THF. In the rabbit preliminary evidence is Heritability of Resistance now available to show that the appendix may act as such a site (2). The genetic basis of resistance in C57BL/Ka mice to tumor The nature of THF and of its mechanism of action in endowing induction by polyoma virus was investigated by testing the lymphoid cells (IP) with immunologic competence (IM) remain ability to resist virus in the segregating populations derived from to be determined. crosses of C57BL with a highly susceptible strain, C3Hf/BiLw. All mice delivered of Polyoma HI-negative mothers were inocu Role of the Thymus in Oncogenesis lated at birth with a thymotrophic strain of virus P-388 D1 (9). Thymic epithelial neoplasms and pleomorphic neoplasms of the There has been intermittent interest for years in the possibility salivary glands were the common morphologic forms observed that immunologic processes play a part in the induction or in this group of mice. Chart 9 shows the cumulative frequencies repression of neoplasms. It is only fairly recently that this possi observed in these resistant and susceptible inbred strains, the bility has been adequately put to experimental test. F1 mice (including reciprocal crosses), and backcross mice to the Important advances in 2 areas have contributed significantly resistant (F1 x BL) and to the susceptible parents (F1 x C3H). to such studies: (a) the conclusive demonstration that a variety All C3H mice developed neoplasms by 6 months; none of 52 of primary as well as transplantable neoplasms possess â€œnewâ€•C57BL mice were tumorous throughout a 14-month observation antigens capable of eliciting specific immune responses; and (b) period. The results from the F1 and backcross generations The demonstration that neonatal thymectomy considerably strongly suggest that a single (or a few) gene is the major deter reduces those immunologic responses broadly classified as cellular minant of resistance to polyoma tumor oncogenesis. In the histocompatibility reactions. absence of definitive progeny tests an equivocal judgment cannot The impairment of immunologic competence observed in most be made, however. Single genes are known to determine host strains of mice subjected to thymic removal in the neonatal susceptibility or resistance to certain other viruses (4, 59, 93). period has been described above. The immunologic deficits Exactly how the determinant gene (genes) functions is not known; extend to humoral antibody formation, delayed-type hyper however, C57BL strain mice become immunologically competent sensitivity, and homograft rejection. There is not, however, a soon after birth before other inbred strains and are known to be generalized immunologic deficiency since the capacity to respond relatively resistant to the induction of tolerance (6, 76). to some antigens is unaffected. The ability to reject allogeneic Recently Rubin (91) has reported that susceptibility to pseudo grafts of normal or neoplastic tissue appears to be generally in types of RSV is determined by a single pair of autosomal genes fluenced, especially when antigenic differences between recipient and that tumor induction is determined by the responsiveness of

560 CANCER RESEARCH VOL. 26

HERITABILITY OF C57BL RESISTANCE TABLE 10 TO POLYOMA VIRUS GROWTH OF POLYOMA TUMOR 4198a IN (Bl x C3H)F1 MICEâ€”THYMECTOMIZED AND INTACT LITTERMATES

mice positive/ U) TreatmentNo. Polyoma titer1. No. challengedAv. HI U) 4 -J 0. Nonthymectomized + virus (22%) 0 (U 2. Thymectomized + virus 42/49 (86%) 1150 z 3. Nonthymectomized + Tris' z buffer 25/26 (96%)1350 <100 (U U Thymectomized + Tris buffer15/69

Lii 0. a Tumor 4198 arose in tissue-cultured cells obtained from sub (U > cutaneous tissues of a C3Hf/HeN mouse. Polyoma virus was added to the culture and transformation occurred after 16 weeks. -j These neoplastic cells have been maintained in vitro and in vivo since 2/2/61 and new antigen has persisted. The in vitro cultured

U line was used for the above inoculations. Thymectomies were done at 3 days of age. Numerous and exhaustive tests for detection of polyoma virus have been made and all were negative. The 0 2 4 6 8 2 15 challenging dosewas 2 X 10@4198tumor cells. LATENT PERIOD (months) b Tris, tris(hydroxymethyl)aminomethane. CHART 9. Cumulative incidence of polyoma-type neoplasms in resistant C57BL/Ka, susceptible C3Hf/Bi mice, their F1 hybrids antigen, it quickly and effectively rejects the cells. This capacity (both C311 x C57BL and C57BL x C3H) and backcrosses of F1 to of immunized adult mice to reject neoplasms which ordinarily resistant and to sensitive parents. Numbers in parenthesis = grow progressively and kill the host is known not to be related to total number of mice observed throughout 15 months. P-388 D1 circulating antiviral antibodies. Recent observations in this strain of polyoma virus injected at birth. laboratory (R. C. Ting, Effect of Thymectomy on Transplanta tion Resistance Induced by Polyoma Tumor Homografts) show the target cells ; susceptibility and resistance are therefore ex that immunization of adult C57BL mice with a virus-free poly pressed in vitro as well as in the live chicken, in contrast to oma tumor containing new cellular antigen is as effective as C57BL response to polyoma virus. immunization with virus in achieving refractoriness to a subse quently grafted syngeneic (C57BL) polyoma-type neoplasm. Antigenicity of Polyoma Tumors That this specific transplantation resistance is due to an immunologic reaction which is mediated mainly by lymphoid There is good evidence that neoplasms arising after infection cells is shown in the experiments using thymectomized mice with polyoma virus have specific antigenicity (16, 31, 96, 99). This which have received an immunizing dose of polyoma virus â€œnewâ€•antigenapparently is similar in mice from tumor to tumor (Table 10, Group 2). These adult mice are incapable of resisting and from strain of virus to strain of virus, although preliminary syngeneic tumor cells despite the presence of high levels of evidence from 2 laboratories now suggests qualitatively different antiviral antibody. Restoration of thymectomized mice by s.c. antigens induced by the LID1 strain and the SE 3049 strain of thymic grafts is known to restore the capacity to resist tumor polyoma virus (35, 98). Adult mice that have been given virus challenge (99). in an immunizing dose are known to be subsequently resistant to transplantation of syngeneic polyoma type tumors but not to The Thymus and Carcinogenesis other neoplasms. This phenomenon is best illustrated in Table 10; compare Groups 1 and 3. Those mice given an inapparent It is now apparent, as mentioned earlier, that many neoplastic infection with the LID1 strain of polyoma virus 3 weeks prior to growths contain â€œtumorâ€•antigens@ not present in the host challenge with the polyoma-induced tumor, 4198, resist challenge 3 It should be noted here that the antigen or antigens discussed with 2 x 1O@cells, whereas 25 of 26 noninfected mice grew the are those capable of evoking a homograft-type reaction. They are neoplasm progressively with death resulting in 3â€”4weeks. demonstrable only by resistance to syngeneic tumor challenge in The refractoriness of virus-immune adult mice to transpianta animals immunized with tumor cells containing the antigen or by tion of syngeneic polyoma-induced tumors may be explained on the specific virus. Such â€œtumorâ€•antigens have also been demon the basis of new virus-specific cellular antigens in the trans strated in other DNA tumor-inducing viruses such as SV-40 and formed cells. Habel (31) and Sjogren et al. (96) 1st developed Adenovirus-12 (15, 33, 44, 101). More recently it was shown that 1 or more new specific antigens (â€œneoâ€•antigens)present in certain this concept. Upon virus inoculation of adult mice, normal cells virus-free tumors, as well as in nonneoplastic cells infected in are in fact transformed to clones of neoplastic cells. The immuno vitro, react with sera from animals carrying transplanted tumors, logically competent adult recognizes the new cellular antigen as as determined by complement fixation (CF) reactions (8, 36). The â€œforeignâ€•andis capable of rejecting these cells in a homograft precise relation of the â€œneoâ€•antigensto â€œtumorâ€•antigens is not type reaction. The animal becomes sensitized to new antigen, known. Recently, however, Habel (32) has shown that the polyoma and when later challenged with tumor cells containing the same virus tumor antigen and CF antigens in polyoma-type tumors are

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TABLE 11 PoLY0MA-INDucED NEOPLASMS IN SEVERAL STRAINS OF MICE FOLLOWING EARLY THYMECTOMY

No. Virus A@eat virus @ Strain Group Virus strain Concentration infection No. of mice with Mean latent period @ litters (pFU)a (days) tumors/No. infected (Months)

@ C571M Thymectomized (3 day) 8 LID-iâ€• 2 X 10@ 4-7 18/26 (73%) 24 (2-44) @ Control 0/15 Thymectomized (3 day) 15 LID-i 2 X 106 14â€”18 21/42 (50%) 3 (24â€”4) @ Control 0/39 @ Thymectomized (1 day) 10 LID-i 2 X 106 4_7 15/19 (78%) 2 (14â€”3) @ Control 1/37 44

@ A Thymectomized (1â€”5day) 12 LID-i 2 X 106 4-6 14/33' (43%) â€˜q1 1 Control 0/26

(ill x C3H)F1 Thymectomized (3 day) 4 LID-i 2 X 106 10â€”15 3/11 (28%) 34 (4â€”5) Control 0/8 @ Thymectomized (3 day) 7 LID-i 4 X 10@ 16â€”30 4/25 (16%) 54 (24â€”114) Control 0/15 @ Thymectomized (1 day) 6 LID-i 2Xi0@ 5â€”6 8/10 (80%) 34 (2â€”54) Control 0/16

@ C3Hf/Lw Thymectomized (3 day) 5 LID-i 2 X 10@ 7-16 9/15 (60%) 54 (4â€”74) Control 0/10 @ Thymectomized (3 day) 7 S/M i x 10@ 4/13 6/21 (29%) 94 (84â€”114) Control 0/15

C3Hf/Bi Thymectomized (3 day) 5 LID-i 2 X i06 8-14 16/19 (84%) 34 (2â€”i2) Control 2/10 54 (4â€”7)

a PFU , plaque-forming unit.

. For a description of LII)-i virus, see Ting and Law (99).

C The numbers in parentheses give the range. d Multiple neoplasms. See the text. a ljntisual polyoma-type neoplasms within 4-month period. See the text.

tissues. This applies particularly to those tumors induced neoplasms resulting from introduction of polyoma virus and the by certain tumor viruses, discussed above, and by chemical mammary tumor virus and in relation to methylcholanthrene carcinogens (43, 78, 86). In time this condition may hopefully be (MCA)-induced neopla.sms. found to be true of other neoplasms. There is suggestive evidence Table 11 shows the results of tumor induction by polyoma that the lymphomas induced by several of the leukemogenic virus in several strains of mice normally susceptible or resistant viruses contain â€œnewâ€•antigenswhich have immunologic signifi to the oncogenic effects of polyoma virus. A high frequency of canee in homograft reactions (42, 84). It should be noted, how neoplasms was observed in all Strains thymectomized at 3 days ever, that the leukemic cells studied in these systems still contain of age, particularly if virus was given within the 1st week of life. virus. The common type of neoplasm observed in the refractory It is now also apparent that immunologic inadequacy facili C57BL, C3Hf/Lw, and F1 mice was of the parotid gland, usually tates carcinogenesis in certain experimental situations. These bilateral; other salivary glands were neopla.stic to varying extents. findings imply that antigenic neoplastic cells may periodically A strain of mice which are highly refractory, however, showed arise in the experimental animal and in man but are promptly neoplasms of the mammary gland and hair follicle as well as eliminated or suppressed by the host's adequate immunologic parotid neopla.sms. The highest frequency and multiplicity of mechanisms (10). polyoma-type tumors was observed in the normally susceptible In the sub@quent discussion an attempt is made to evaluate C3Hf/Bi strain of mice. Here, although LID-i virus was given our l)resent knowledge of the relationship of immunologic com late, at 8â€”14days of age, multiple neopla.sms were observed in petence to carcinogenesis initiated by several agents. The role of most 3-day thymectomized mice. In addition to salivary glands, neonatal thyniectomy which leads to an assortment of immuno the sites involved were mammary glands, hair follicles, s.c. logic deficiencies has been studied in relation to virally induced connective tissues, kidney, and bone. The response to polyoma virus of C57BL and F1 mice thymoc not the same; the presence of demonstrable anti-tumor CF anti tomized within 24 hr of birth is shown for comparison in Table 11. bodies is siot necessary for the immunized animal to resist chal The period of sensitivity of salivary gland tissue to the onco lenge of a syngeneic polyoma tumor. Neither of these antigens genic effects of LID-i polyoma virus in C57BL thymectomized represents or is related to viral antigen. mice extended to at least 30 days of age, the latest age at which

562 CANCER RESEARCH VOL. 26

TABLE i2 RESPONSE OF THYMECTOMIZED AND RESTORED C57BL MICE TO LID-i STRAIN OF POLYOMA VIRUS

with tumors/ latent SA.Thymectomized Grou Cmark pNo. littersNo. No. miceMean period (mo)R

at 3 days (65%) (2_4)a polyoma virus injected s.c. at 13â€”i8days PFUb/mouseB.ThymectomyThymectomy + dissociated 2/29 (7%) 5, 7LID-i 4 X i0@ spleen cells Sham and intactc2i20/31 0/4224

at iâ€”3days (38%) (3â€”12) virus injected sub taneously at 20-30 days PFU/mouseC.Thymectomy 4 X 10@ Thymectomy + Millipore 1/8 (12.5%) 14Polyoma chambers containing neo natal thymus at i4 days Intacti29/24 0/206

at i day (75%) (2â€”4) virus given at 14 days 2 X 106 PFU/mouse Thymectomy + syngeneic 2/12' (i7%) 34, 9Polyoma thymic graft at i day Intact79/i2d 0/3024

a Numbers in parentheses give the range. b PFU, plaque-forming unit.

C No differences in response between sham-operated and intact mice; other controls in Groups B and C were therefore not given a sham operation. d Two mice died of â€œwastingâ€• before tumor induction.

e One animal died â€œwastedâ€• but with a parotid gland neoplasm at 34 months of age. virus was introduced in the present experiment (Table i2, newborn thymic tissue, (b) i.v. introduction of adult lymphoid Chart iO). cells from syngeneic donors, or by (c) i.p. installation of cell-tight Several variant â€œstrainsâ€•of polyoma virus, in addition to Millipore diffusion chambers containing neonatal thymic tissue LID1 (52) have been effective in inducing polyoma-type neo (13, 49, 70, 83). plasms in 3-day thymectomized mice. S/M, a relatively non It has been shown (46) that C57BL mice, thymectomized at oncogenic small plaque variant, induced parotid gland tumors birth, were restored if given 6â€”12x i0@dissociated syngeneic in C3Hf/Lw mice although the latent period was lengthened over spleen cells from the adult 48 hr after birth. The restored mice that observed with the LID-i strain (see Table ii). SE 3049 and had a normal growth pattern, there was no depletion in lymphoid KP polyoma virus strains induced salivary gland neoplasms organs, and they had a normal capacity to form hemolysins to and ameloblastomas of the gingival epithelium, respectively, sheep erythrocytes. In addition, only 36.6% of the restored in thymectomized C57BL mice. No tumors have been observed C57BL mice had parotid gland tumors; 80% of the neonatally in C57BL intacts inoculated at birth with either of these latter thymectomized group were tumorous. In this group polyoma or with other variant strains. Interestingly, Malmgren et al. virus was administered early, at 4â€”6days of age, that is 2â€”4days (60) observed an increased frequency of polyoma-induced neo after inoculation of adult spleen cells. If virus is introduced later, plasms in thymectomized mice infected early in life with a known at 8â€”lidays after the introduction of 20 x i0@syngeneic spleen â€œattenuatedâ€•strain of polyoma (M polyoma). cells, a return to nearly complete refractoriness is observed (see Table i3). Adult syngeneic cells from lymph nodes are also Oncogenesis in Thymectomized Mice Restored by Several shown to be nearly as effective as spleen cells, but spleen cells Methods from neonatally thymectomized mice are less capable of restoring the capacity to resist polyoma virus oncogenesis. Evidence is now presented to show that interference with the It will be seen in Table 12 that other methods of restoration immune mechanism(s) is a necessary prerequisite for successful of neonatally thymectomized mice also effectively prevented the tumor induction in normally refractory neonatal mice and in induction of the usual polyoma-type neoplasms encountered in refractory adults. thymectomized mice. The immunologic defects resulting from thymectomy within In Group A, splenic cells from adult syngeneic donors were 24 hr of birth, the lymphoid cell depletion, and the disturbed effective whether given i.v., 6â€”i2x l0@cells at 4 days of age, or growth (resulting in â€œwastingâ€•insome but not all inbred strains i.p., 3 x i0@cells at 7 and again at 9 days. The results of both of mice) can be prevented by several methods: (a) grafting of groups were similar and are combined. In this group of mice

APRIL i966 563

AGE RESPONSE TO POLYOMA VIRUS C57 BL THYMECTOMIZED AND RESTORED 3â€”DAY THYMECTOMY POLYOMA VIRUS (2â€”4XIO6PFU) 100-@jOO.

U /4 days 0 0 a a /3â€”/adoys 21t mo. U) U) U) U) 4 4 0. a@ 0 0@75- (U 20-30 days [email protected]

0).- 0Ii

C.) 2! C.) z (U00 (UZ25->. (UIiiLi.Li. L@@: T T+S C I T+MC C T T+G C @ I I I _O 10 20 30 40 CHART 11. Frequency of parotid gland neoplasms in the 3 groups AGE AT VIRUS INFECTION (doys) of 3-day thymectomized C57BL mice restored by several methods. T, thymectomy; C, intact ; T + 8, thymectomized mice restored CHART 10. Frequency of parotid gland tumors in C57BL strain with adult syngeneic spleen cells; T + MC, thymectomized mice mice thymectomized at 3 days of age. Polyoma virus, 2â€”4X 10@ bearing Millipore diffusion chambers with thymic tissue ; T + G, plaque-forming units, injected at times after birth indicated by thymectomized mice bearing syngeneic thymic grafts. Age in triangles. Average latent periods are shown in figures above each days at which LID-i polyoma virus injected is given above each point. group.

TABLE 13 TABLE 14 EFFECT OF THYMECTOMY AT BIRTH AND LYMPHOID INHIBITION OF POLYOMA-TYPE NEOPLASMS IN THYMECTOMIZED CELL REsToa@TIoN ON THE INDUCTION OF MICE INOCULATED WITH LYMPHOID CELLS AFTER NEOPLASM5 IN C57 BLACK MICE POLYOMA VIRUS INFECTION with GroupNo. miceNo. parotid period latent tumorTimorousLatent of (mo)Thymectomy Experimental groupNo. miceNo.withneoplasmsFrequencyneoplasms (mo)Thymectomy (%)Av.

Thymectomy + spleen 45 2 4 4 + lymphoid cells cells given at: Thymectomy + lymph 21 3 i4 3 1. 3-fidays node cells 2. 8â€”12days 21 3 14 4.5 Thymectomy + spleen 28 8 293 3.5 3. 14â€”21days 10 4 40 3 cells from thymectomized Thymectomy only 29 18 622.5 3 donors Intactii 301 09 Intact43 2629 068 Thymectomy of C57BL mice at 3 days of age; polyoma virus, 20 X iO' adult cells of each type given intravenously 24 hours LID-i strain, injected s.c. at 6â€”8days of age at a concentration after thymectomy. of 2 X 10 plaque-forming units/mouse. Adult syngeneic lymphoid Polyoma virus, 2 X 10@PFU given 8-u days after introduction cells (spleen) were inoculated i.v. at the time indicated in Groups of adult lymphoid cells. 1, 2, and 3 after polyoma virus infection. Weight-adjusted doses The usual polyoma-type tumor in C57BL mice involves a single of lymphoid cells were inoculated at 2 X 10Â°cells/gm of body or both parotid salivary glands; other salivary glands were neo weight. All neoplasms were of parotid gland origin and involved plastic to varying extents. to varying extents the other salivary glands.

thymectomized at 3 days of age, the usual stigmata associated Millipore chambers were recovered 12 months from the with thymectomy performed within 24 hr of birth were not restored, tumor-negative mice in Group B. In these, the mass of observed. There was little or no evidence of â€œwasting,â€•theredid persisting tissue was principally necrotic, but in many viable not occur in general lymphoid depletion in organs or peripheral tissue was observed in the form of fibroblasts and nests of reticu blood, and hemolysin titers to sheep erythrocytes were similar lar epithelial tissue. to those of intact littermates; 3-day thymectomized C57BL Chart 11 shows the relative effects of restoration by the 3 mice were also capable of rejecting allogeneic skin grafts. Thy methods employed. mectomy at this time still has an effect on the development of Preliminary data obtained in this laboratory also show that immunologic reactivity to certain antigenic stimuli (71). adult syngeneic lymphoid cells are effective in preventing the

564 CANCER RESEARCH VOL. 26

w D (I.) (I) F- IL 0 E O :@ IL. 0.

TIME IN DAYS

CHART 12. Growth curves for LID-i strain of polyoma virus in intact and 3-day thymectomized C57BL mice. Vi r@ts injected (1 X 10 plaque-forming unit) at 7 days of age. Mice in each group were sacrificed at various times from 4 hr to 18 days for determination of virus concentration. Points represent average concentrations for each of 3 tissues: kidney, liver, and salivary glands (3 mice for each point). Virus assays were done according to the method of Dulbecco and Freeman (20). Hemagglutination-inhibiting antibodies deter mined from Day 4 to Day 30 in similar groups were nearly identical. induction of neoplasms even if these cells are administered i.v. nificant differences were found in Polyoma HI antibody titers, (2 x 10@cells/gm of body weight) as late as 12 days, but ap determined periodically from Day 4 to Day 30. These results re parently not at 14â€”21days after infection with polyoma virus lating to virus growth and antibody formation are similar to the (see Table 14). Previously sensitized lymphoid cells appear to be results observed in thymectomized and intact Swiss-Webster mice more effective in inhibiting the induction of these neopla.sms in their response to LCM infection (57), except that in the latter (E. Leckband, R. C. Ting, and L. W. Law, unpublished data). experimental situation thymectomized mice were more refrac tory to the lethal effects of LCM than their intact littermates. Virus Growth and Antibody Formation in Thymectomized Virus growth patterns and antibody formation, however, were Mice similar in thymectomized and intact mice.

It is unlikely that the striking increase in susceptibility to â€œSpontaneousâ€• Infection In Thymectomized Mice polyoma virus oncogenesis after thymectomy is the result of Leading to Tumor Induction nonspecific factors affecting the general health of the mice; those mice thymectomized at 3 days of age were in excellent condition The studies recorded here were initiated to examine the effect showed no evidence of the trophic disturbances characteristically of early thymectomy on the development of spontaneous mam seen in these strains of mice thymectomized on the day of birth. mary cancer in the females of a high mammary tumor strain and Conceivably, the absence of thymic tissue could enhance virus at the same time to investigate the effect of thymic removal on growth leading to an earlier and greater concentration of virus methylcholanthrene-induced fibrosarcomas in their male litter in the target organs. The highly oncogenic S polyoma virus, for mates (47). example, showed a 1000-fold greater titer at 5 days after early C3H/HeN strain mice from the NIH colony were used. These infection than did the M polyoma, low-oncogenic variant (25). mice carry the mammary tumor milk agent. They were thymec It is clear, however, from the results shown in Chart 12 of the tomized at 3 days of age. Completeness of thymectomy was virus growth curves in thymectomized and intact C57BL mice checked by serial sections of tissue obtained at necropsy from the that the virus growth pattern is not a significant factor in in upper mediastinal area. At least 2 members of each litter were creased susceptibility of neonatally thymectomized mice; no sig left as intact controls. Litters were raised by their own mothers

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TABLE 15 POLYOMA-TYPE NEOPLASMS AND MAMMARY ADENOCARCINOMA IN VIRGIN C3H/HeN FEMALE MICE: 3-DAY THYMECTOMY AND ROOM INFECTION

HI ANTIBODIES (AccoamNc TO LITrES)No. Gsou@POLYOMA WITS NEOPLASUS/ (mo)+â€”TiterThymectomized No. MICELATENT PERIOD

(4@i1)bThymectomized (65.2%) <1:100 0/34

15/57 (26.3%)

Intact6 69 1:600 0/15 91:4500' <1:16015/23 0/i6

0/316.8

adenocarcinomas (45.2%) (1O-i6) IntactMammary show no relationship to 21/31 (67.7%)i2.7 10.6 (8â€”16) Polyoma HI antibodies19/42

a The higher average titer among thymectomized mice resulted from excessively high titers in several tumorous mice. If these are excluded, average titers are similar in thymectomized and intact mice. b Numbers in parentheses indicate the range.

and at 4â€”5weeks were separated according to sex, but with 1 male mouse developed parotid gland neoplasms, and this was thymectomized and intact mice remaining together, 6â€”8mice/ at 6 months of age, although more than 4 had evidence of poly cage. These mice were housed in an animal room containing oma infection. other experimental mice, some of which had received polyoma Mammary tumors are now appearing in the remaining C311 virus parenterally. Females received no further treatment ; males females, beyond 74 months of age, and these have the typical received 0.1 mg of 3-methylcholanthrene (MCA) dissolved in morphology of such neoplasms arising in mice with the Bittner 0.2 ml tricaprylin, s.c., at 8 weeks of age. The mice were examined milk agent, reproducing mammary semi in many areas and for daily for neoplastic growths. Unexpectedly an untreated female the most part classified as adenocarcinomas. Histologic compari developed bilateral parotid gland tumors when 44 months old. son of these adenocarcinomas with a typical parotid gland neo At this time Polyoma HI antibodies (22) were determined in the plasm is shown in Chart 13. The mammary tumors occurred sera of both males and females. The members of 6 out of 15 litters both in Polyoma HI-positive and HI-negative females. After a of the untreated females showed HI antibodies ranging from 16 months' observation the frequency is lower and the latent 1 :400 to 1 : 14,800. An HI antibody survey taken when the mice period longer among the thymectomized mice (Table 15). At were 2 months of age showed all to have titers of < 1 : iOO (HI i year there was a pronounced difference in frequencies: 7/42 negative). Later, 14 additional females presented polyoma-type (i7%) in thymectomized females and 21/31 (52%) in intact neoplasms but only those among the 23 thymectomized mice females. These findings are similar to those reported among comprising the 6 HI-positive litters. All intact mice, showing breeding females of another C3H subline (61). nevertheless polyoma virus infection, remained tumor free, as The pattern of spontaneous polyoma virus infection in our did all members of the 9 HI-negative litters (see Table 15). inbred mouse colonies in which experimentally inoculated mice The neoplasms, grossly, were confined to 1 or both parotid were housed has been reported (89). A unique feature of the spon glands. Histologic examination, however, also revealed numerous taneous infection pattern was the nearly complete absence of neoplastic area.s in other salivary glands. The histologic appear virus, as determined by HI antibody, up to 2â€”3months of age ance of these neopla.sms was similar to that described earlier (48). but increasing in frequency with age beyond 3 months; although There were poorly defined tubular structures with a tendency a high frequency of polyoma virus infection was observed in some toward loss of epithelial character and gain of spindle-cell strains of mice, the occurrence of polyoma-type tumors was rare. features. Table 16 compares the infection rate in C3H mice for 1959 and No differences were observed in the frequency or latent period those mice used in the present study. of MCA-induced fibrosarcomas between 3-day thymectomized Probably the only way in which the mouse, infected through males and their intact littermates. Eighty-one % (47/58) of natural means as an adult, can develop a polyoma-induced neo thymectomized mice and 79% of the controls (23/29) had neo plasm is by the chance occurrence of an event which reduces its plasms; the average latent periods (after carcinogen introduction) immunologic competence. Thymectomy early in life is an effec were 76 and 74 days, respectively. Among the 3-day thymecto tive method of accomplishing this and is apparently more effec mized C3H/HeN males, only 23 were at risk to polyoma virus tive than total body irradiation. C3H mice in our same colony infection from 44 to 64 months, during part of the period when which received either 300 r or 150 r X 4, 80% of which had high parotid gland tumors appeared in their female littermates; only levels of HI antibodies, have failed to develop polyoma-type neo

566 CANCER RESEARCH VOL. 26

0 Thym (35) malignant cells that may arise from time to time throughout the . Intact (3/) life of higher organisms. Our results with virus-induced mammary cancer and with MCA-induced fibrosarcomas were indeed un 0 Thym (4/).\ @1 expected in view of the evidence that both types of neopla.sms I In/ad (/6) J contain â€œtumor-specificâ€•antigens (43, 86, 104) that should be 75 - beneficial to the host in evoking homograft reactions. Since the results of 2 groups of investigators (16, 29) gave sug gestive evidence of an effect of neonatal thymectomy on shorten ing the latent period and influencing the frequency of MCA 50 - induced fibrosarcoma, our earlier experiments were rel)eated in both C3H and C57BL strain mice thymectomized at 3 days of age. The dose of MCA was reduced to 50 pg/mouse. There were found no differences in the latent periods, frequency of induced 25 - neoplasms, or growth potential of the induced neoplasms in thymectomized and sham-thymectomized littermates. The

I results in C3H males and females are shown in Chart 13. â€˜I -n 0 30 60 90 I2O 150 Thymectomy and Leukemogenesis LATENT PERIOD (days)

CHART i3. Cumulative frequencies of fibrosarcomas in C3H/ There are a multitude of very interesting associations between HeN males and females following injection of 50 j@gof MCA at 6â€”8 diseases in man and thymic lesions or abnormalities. Some of weeks of age. Latent period = time following introduction of car these are as follows (see Ref. 26): cinogen. See the text for frequencies and latent periods of C3H 1. The simultaneous development of acquired agammaglohu males in a separate group but given a higher concentration of linemia and benign thymoma. MCA. Numbers in parenthesis = total numbers in each group, 2. Extreme deficiency of thymic development in the Swiss type intact and thymectomized. of agammaglobulinemia. 3. Many patients with ataxia telangiectasia lack some im TABLE 16 munoglobulins and are immunologically defective. Many, how INCIDENCE OF HI ANTIBODY TO POLYOMA VIRUS ever, have normal immunoglobulins and normal numbers of (ROOM INFECTION) plasma cells. 4. Many patients with myasthenia gravis have demonstrable GROUP1-3 INCIDENCE BY AGE morphologic abnormalities of the thymus. GROUPANTIBODY mo.No.mo4â€”8 mo@lO 5. There is some suggestion of an increased risk of leukemia in children with congenital agammaglobulinemia. %1959â€”Colony %No. %No. There are many other interesting associations, but it should be borne in mind that these are suggestive a.ssociations; the evi 4 27 46 dence is not conclusive that there is a causal relationship between 1964_C3HB4/95 0/40 061/23023/53 4488/19228/53 53 any of these diseases and thymic dysfunction.

a Includes a sample of both males and females used in the On the other hand it is shown that 1 form of lymphocytic leu present study. At 12 months the HI frequency of the remaining kemia in the mouse is thymic dependent. The earliest observa mice had reached nearly 70% . All except i parotid tumor, how tions were made by McEndy et al. (63) who showed that ever, appeared before 9 months. thymectomy in young adults prevented the development of lym phocytic leukemia in high leukemic AKR mice. It was shown plasms. C3H mice, thymectomized within 24 hr of birth, but subsequently that thymectomy in young adults also l)revented restored with adult syngeneic lymphoid cells (100) and housed the induction of thymic lymphomas in mice by whole body ir in the same laboratories have remained nontumorous; 31 such radiation (41), chemical carcinogens (50), and leukemogenic C3H mice reconstituted with spleen cells, lymph node cells, or viruses (30, 58, 68). bone marrow were in this group and all had high levels of Poly Thymectomy does not prevent the development of the disease oma HI antibody when tested at 9â€”12months of age. simply by removing thymic cells which are presumably most These experimental results are also in accord with the concept susceptible to neoplastic conversion. As shown by Law and that the basis of resistance to polyoma virus oncogenesis is a Potter (5i) and Carnes et al. (11), in thymectomized animals cellular immunity of the homograft type directed against virus bearing genetically identifiable thymic grafts the leukemia.s specific new â€œtumorâ€•antigen contained in the neoplastic cells. which developed were often of host origin and did not originate In the immunologically disturbed animal these antigens are not from cells of thymic tissue. These findings suggest that the non capable of provoking an effective immunologic attack on the lymphoid elements of the thymus (epithelial and reticular cells) cells transformed to neoplasia by virus. These clones of cells are responsible for influencing the conversion of normal to leu therefore grow progressively into frank neoplasms. kemic cells within the environment of thymic tissue. It remains to be determined how important a part this defense Surgical removal of the thymus in 3-month-old potentially mechanism, homograft rejection, plays in the elimination of leukemic mice is known to be as effective in preventing lympho

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TABLE i7 plausible explanation for resistance to polyoma virus oncogenesis LYMPHOCYTIC LEUKEMIA IN C3Hf5/Lw NEONATALLY is related directly to the immunologic capacity of the host animal; THYMECTOMIZED MICE RESTORED WITH ADULT cellular immunity of the homograft type invoked against virus SYNGENEIC CELLS specific â€œtumorâ€•antigencontained in neoplastic cells is involved. In the immunologically disturbed animal (following neonatal leukemic/ age (mo) at deathIntact GroupNo. total No.Mean leukemic thymectomy) these â€œtumorâ€•antigens are not capable of invok ing an effective immunologic attack on clones of cells transformed (74%) (3@i0)a to neoplasia by polyoma virus. Neoplastic clones therefore grow Thymectomized and 4/24b (17%)6.3 li.0 (8â€”13) progressively into frank neopla.sms. In the immunologically corn restored17/23 petent animal, either intact or restored neoplastic conversion occurs, but such cells are aborted by a successful homograft-type a The numbers in parenthesis give the range. reaction. Immunologic restoration was accomplished in the b3/4 Granulocytic leukemia. thymectomized mouse by all 3 of the methods attempted to the extent of permitting adequate homograft rejection and thus cytic leukemia as thymectomy performed in the neonatal period. repressing the origin of neoplastic cells. C57BL mice are known These observations suggested that impairment of immunologic to become immunologically competent soon after birth (76). function of the host animal against probable new specific induced The immunologic deficit resulting from thymectomy at 3 days antigens of leukemic cells (42) was not a major factor in the con of age which allows tumor induction by polyoma virus is indeed trol of leukemogenesis. Recent interesting results have been quite subtle and not easily recognized grossly. Little or no reported by 2 groups (18, 85) which require a reassessment of lymphoid cell depletion was noted and these mice (C3H and the role of the thymus in leukemogenesis at the immunologic C57BL) were quite capable of rejecting allogeneic skin grafts level. These authors found a reduced responsiveness to bacterio and of forming in normal fashion hemolysins and agglutinins to phage T2 and disturbed homograft rejection in C3H mice inocu sheep erythrocytes and HI antibodies to polyoma virus. C57BL lated with Passage A virus. The situation is not simple and not female mice thymectomized at 3 days, however, were less capable easily resolved since the organs responsible for immunologic of rejecting male skin than their intact littermates. Twelve reactivity of the host are directly involved in leukemogenesis. thymectomized females rejected male skin at an average time of Certain observations from this laboratory relate to this point. 68 days, whereas all 15 intact control littermates had rejected Mice of our C3Hf*/Lw high-leukemic substrain (the asterisk skin grafts by 30 days. distinguishes this strain from the normally low-leukemic C3H These results suggest that. the polyoma virus-specific â€œtumorâ€• strain), established through parenteral introduction of Moloney antigen is functionally similar to a minor histocompatibility leukemogenic virus (MLV) and maintained by passage of virus antigen. through the mother's milk (45) have a high frequency of lympho It should be reemphasized that resistance and susceptibility to cytic neoplasms (Table 17). Thymectomy at i month or in the tumor induction by polyoma virus are unrelated to viral growth neonatal period practically eliminates lymphocytic neoplasms. Of patterns and to humoral antibody production to virus. The pat @ 127 C3H mice thymectomized in the neonatal period only 4 of terns were nearly identical in intact and thymectomized C57BL those completely thymectomized developed lymphocytic neo mice. These findings also suggest that a difference in interferon plasms ; yet these mice are known to be immunologically deficient production is not a major factor in increased suscel)tibility to in response to polyoma virus (see above) and in homograft reac polyoma virus. @ tions against certain allogeneic neoplastic tissues. If C3H mice Thymic tissue per se is a necessary antecedent of resistance to are thymectomized within 24 hr of birth but restored with syn polyoma virus tumor induction to the extent that it provides geneic adult lymphoid cells (100) their potentiality to develop maturation of immunologic faculties and consequently prevents lymphocytic neopla.sms is not restored. These results show (a) the progression of neopla.stic clones of cells induced by virus. If that in the absence of thymic tissue the immunologic status of many tumors contain â€œtumorâ€•antigens foreign to the host, it is the host animal is not a major factor in leukemogenesis in this likely that the host can respond immunologically against these species and (b) that immune processes do not play any positive antigens and destroy or inhibit emerging clones of neoplastic part in the production of this malignant disease. cells. Neonatal thymectomy should therefore induce an increased susceptibility to the development of such tumors. The generality Discussion and Summary of the influence of thymectomy remains to be determined. Early The genetically controlled variation in mice to polyoma virus thymectomy in the rat and hamster is associated with an in tumor induction which appears to be determined by 1 or a few creased sensitivity to iolyoma virus (16, 102). Neonatal thy genes is not a property of individual target cells or of individual mectomy in the Syrian hamster did not influence induction by cells which influence viral growth patterns. It thus differs from RSV of gliomas or of fibrosarcomas (G. Rabotti and L. W. Law, the susceptibility and resistance to virus infection studied by unpublished observation), nor was there observed an increased Bang and Warwick (4), Goodman and Koprowski (27), and yield or lowered latent period in mammary carcinoma induced Rubin (91). As mentioned previously, isolated organ cultures of by the Bittner virus or in MCA-induced fibrosarcomas as C57BL mice transform readily to neoplasia in the presence of revealed in this study. polyoma virus ; resistance is therefore a characteristic of the Early in the discussions of polyoma virus tumor induction it intact animal. was pointed out that the striking sensitivity of newborn animals It is clear from the facts presented in this study that the most may be related to the fact that the most responsive tissues were

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FIG. 1. A, Spleen of neonatally thymectomized C3H mouse killed at 8 weeks, showing generalized atrophy and absence of follicles. H & E, X 40. B, Spleen of neonatally thymectomized C3H mouse bearing i.p. implant of syngeneic thymic tissue in Millipore diffusion chamber. Killed at 8 weeks; note well-developed follicles with germinal centers and heavy infiltration of red pulp withlymphoid elements. H & E, x 40.

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FIG. 2. A, Inguinal lymph node of neonatally thymectomized C3H mouse killed at 8 weeks showing striking atrophy and paucity of lymphocytes in both cortex and medulla. H & E, X 56. B, Inguinal lymph node of neonatally thymectomized C3H mouse bearing Millipore diffusion chamber with syngeneic thymic tissue. Installation of chamber at 4 weeks; animal killed at 8 weeks. Architecture of node normal with presence of follicles and many lymphoid elements. H & E, X 50.

APRIL 1966 573

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FIG. 3. A , Mammary tumor in C3H/HeN female sacrificed at 8 months of age. H & E, X 140. B, Parotid gland neoplasm in C3H/HeN female sacrificed at 7 months of age. H & E, X 140.

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Lloyd W. Law

Cancer Res 1966;26:551-574.

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