CELLULAR

IMMUNOLKY

23, 356-375 (1976)

The Role of Thymus Subpopulations

in ‘7”

Leukemia

Development1

R. CHAZAN AND N. HARAN-GHERA Drpartmclrt

of Chemical

Iwzwtz~nology,

The

Weizmam

Received January

Imtitute

of Scicncc, Rclzovot,

Israrl

16,1976

Based on antigenic properties of the cell surface of mouse thymocytes, spontaneous (AKR mice) and induced T leukemias (C57BL/6 mice) were shown to have characteristics of the minor thymus subpopulation, namely, low levels of 8 and high levels of H-2. Leukemogenic agents (fractionated irradiation or inoculation of radiation leukemia virus) were shown to induce a transient or permanent change in thymus population patterns. Within several weeks following leukemogenic treatment there was a relative enrichment of thymocytes bearing low levels of 6 and high levels of H-2 and partially resistant to hydrocor+isone and capable of induceing a graft versus host response. Transplantation bioassays carried out indicated the lack of demonstrable leukemic cells in the thymus within several weeks following the leukemogenic tre’atment. Similar spontaneous age-related changes in the pattern of the nonleukemic AKR thymus, namely, increase in the high H-2 thymus subpopulation and a gradual decrease in the percentage of B-bearing cells was observed from 5 months onwards. The relationship between the availability of certain thymus subpopulations and the ultimate overt leukemia development was indicated.

INTRODUCTION The target organ for the overt development of most spontaneous or induced murine lymphoid leukemias is the thymus. Thymus lymphocytes can be divided into two categories on the basis of surface markers and functional properties. The major thymus subpopulation, considered to be steroid and radiation sensitive, consists of immunologically inactive cells, and its antigenic characteristics include high levels of 6 antigen and low levels of H-2 alloantigen and the TL antigen (when present at all). The minor subpopulation, which is relatively radiation and cortisone resistant, has a high H-2 antigen content and lower levels of 8, lacks the TL antigen, and possesses the capacity to respond to phytohemagglutinin (PHA), mount graft versus host (GVH) reactions as well as reacting in mixed lymphocytes cultures. The experiments to be reported here suggest that most of the spontaneous or induced T leukemias originate from the minor thymus subpopulation, namely, consist of thymocytes bearing high levels of surface H-Z alloantigens and low levels of 8. The different leukemogenic agents change the pattern of the normal thymus subpopulations and thereby provide perhaps the lymphoid population SUSceptible to neoplastic transformation and/or proliferation of the preleukemic cells. 1 This investigation was supported by the United States Public Health Service Research Contract No. l-CB-43930. 356 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

THYhlUS

SUBPOPULATIONS

IN

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3.57

The transient availability of thymocytes bearing high levels of H-2 in abundance for several weeks following the leukemogenic treatment might be a prerequisite for the development of overt T lymphoid leukemia in mice. MATERIALS

AND

METHODS

Mice. C57BL/6, (Balb/c x C57BL/6)F1, AKR/J, and C3H/eb were bred and reared at The Weizmann Institute of Science Breeding Center. They were kept in metal cages at 22°C room temperature and fed chow pellets and tap water. Antisera. Anti-8 C3H serum was prepared by immunizing AKR/J mice with C3H/eb thymocytes taken from 8-lo-week-old mice ( lo7 cells injected intraperitoneally at weekly intervals for 7 weeks). H-2b alloantiserum was obtained following six weekly injections of 5 X lo7 C57BL/6 spleen cells (from S-loweek-old mice) in C3H/eb mice, and anti H-2” serum by 5 x lo7 AKR/J spleen cells in CSH/eb mice (donors and recipients aged S-10 weeks). The immunized mice were bled 1 week after the last cell inoculation. Percentages of &bearing cells and high H-2b-bearing cells were determined by the cytotoxic assay. Cell suspensions of various treated thymuses or leukemias were prepared in cold Tyrode containing 0.1% bovine serum albumin (BSA). The suspensions were washed twice and adjusted to lo6 cells/ml. To 0.1 ml of cell suspension an equal volume of antiserum (1 : 16-l : 32 dilution) was added. The mixtures were incubated for 30 min at 37°C and, after several washings with Tyrode-BSA (O.l%), guinea pig complement (absorbed on agarose and diluted 1: 9 in Kohner’s saline) was added and the cells were further incubated for 30 min at 37°C. The index of cytotoxicity was estimated by the dye exclusion of trypan blue. Inznzlllao~zlorescence. Fluorescein-conjugated goat anti-mouse IgG or IgM sera (purchased from Meloy Laboratories, Springfield, Va.) were used. Direct Iwmno&oYescence for surface IgG-IgM : Cell suspensions (5 x 106) were washed in 10% gelatin in Tyrode, then pelleted down and incubated in 0.5 ml of goat anti-mouse immunoglobulin diluted 1 : 10 in phosphate-buffered saline (PBS) at 37°C for 30 min. Cells were washed three times in 1% gelatin in Tyrode, then resuspended in 50% glycerine in PBS and kept at 4°C until read under LIV light. Indirect inlnzunofluorescence for determination of high H-2 bearing cells : Diluted anti-H-2” antiserum (1 : 16-l : 32) was added to lo6 cells; the mixtures were incubated for 20 min at 37°C and washed several times. After the final washing, the cells were suspended in 0.1 ml of rabbit anti-mouse IgG conjugate and incubated for 30 min at 37°C ; thereafter, cells were washed three times in 1% gelatin in Tyrode and resuspended in 0.5 ml of a solution of 7% BSA, mixed, and centrifuged. The cells were resuspended in a drop of buffered BSA, and smears were prepared on coverslips. The smears were dried and fixed for 10 min in 96% ethanol. Leukewtia induction. The experimental systems used in the present studies involved lymphatic leukemia induction in C57BL/6 mice by exposing &S-week-old C57BL/6 female mice to four weekly doses of 170 R whole body irradiation or by inoculating the radiation leukemia virus directly into the thymus of 6-9week-old mice. The radiation leukemia virus used in the present studies was originally obtained from bone marrow of irradiated nonleukemic C57BL/6 mice and kept by serial passage lines of the virus in thymic tissue of C57BL/6 mice ( 1). In general, such serial passage lines of virus when injected directly into adult C57BL/6 thymus induce a low leukemia incidence (O-20% at an

358

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average latent period of 120 days). The incidence can be increased to SO-loo% by further exposure (within a few days after virus inoculation) of the inoculated mice to a dose of 300-400 R whole body irradiation (2). This type of virus passage is designated in the present study as D-RLV (“D” stands for dependent, since its high leukemogenic effect is dependent on exposing the virus-inoculated mice to X rays). Sometimes, though, a serial passage line of the virus when injected intrathymically into normal C57BL/6 mice would yield a high leukemia without any further exposure to X-rays. Yet, when this incidence (40-100%) virus preparation is injected into the thymus of (Balb/c X C57BL/6) Fr mice its high leukemogenic activity depends on further exposure of the inoculated mice to radiation (acts similarly to D-RLV in C57BL/6 mice (3, 4) ). Such a virus preparation tested in the present analysis was designated as A-RLV (“A” stands for its autonomous high leukemogenic activity independent of the co-leukemogenic effect of X-rays). RESULTS Surface Antigens on Thymocytcs of Spontaneous or Induced Lym@atic

Leukevaias

Since the normal thymus consists of two subpopulations bearing different levels of 6’ and H-2 antigens, (5), it seemed of interest to analyze the surface antigenic characteristics of leukemic thymocytes. The H-2 isoantigen concentration is apparently higher in spleen and lymph node cells than in thymus cells. Winn (6) has shown that isoantigenic receptor sites were distributed in discrete patches rather than uniformly over the entire cell surface, and the number of patches was larger for lymph node cells than for thymus cells. Indeed, lymph node cells were shown to be susceptible to the cytotoxic effect of isoantibodies whereas only 10-15s of thymus cells were affected; namely, H-2 alloantiserum is cytotoxic and lyses only thymocytes having high levels of H-2 antigen. The H-2 alloantiserum and anti-8 C3H serum were used for the evaluation of the surface antigens on thymocytes taken from leukemic AKR/ J mice or from lymphatic leukemias induced in C57BL/6 mice by fractionated irradiation or by RLV (both variants) inoculation. The leukemia incidence obtained by usin,(+ the different induction methods is described in Table 1. The anti-0 and anti-H-2b or -H-2” cytotoxic indexes obtained with the different leukemias (percentage of murine thymocytes begin lysed by 0 and H-2 antiserum in the presence of complement) are summarized in Table 1. All the assays carried out have consistently shown that the leukemias tested were of thymus-derived lymphocyte origin, carrying the specific 6’ surface antigen on the lymphoid leukemic cells (anti-0 killing index being S9-9S%). Although the high H-2 population in the normal thymus consisted of approximately 15% of the total thymus lymphocyte population, the majority of the spontaneous or induced leukemias developin, q in the thymus had the characteristics of this minor cell population. The percentage of thymocytes bearing variable high levels of H-2 alloantigen in the different tested leukemias divided into four categories of incidence is illustrated in Table 1. Among AKR/J spontaneous leukemias, more than half of them were included in the fourth category (60-90s of leukemic thymocytes having high levels of H-2 alloantigen). Among D-RLV plus X-rays-induced leukemias, 80% of the tested mice were classified in category IV and 66% among the fractionated radiation-induced tumors. The most pronounced equal spread

70-100 80-100 70-100

Normal thymus C57BL/6 + 170 R X 4 C57BL/6 + D-RLV

C57BL/6 + D-RLV 3days 400 R C57BL/6 + A-RLV AKR/J, spontaneous 96 92 90

98 87 91

(%I

thymocytes

Mean indexb, &bearing I O-20%

Incidence

wa

g/36 (25%) 2123

(11%)

lo/36 2123

(28%) (8%)

3P-1 (12%)

-

-

2/l@

-

II 21-400/o

(50%)

of viable

were further

7/36 (19%) 13/23 (55Yo)

12/18 (66%) 6/12 (50%) 19/24 (80%)

IV 61-90%

cells in normal

serum)]

exposed to 400 R whole

(22%) 2/24 (8%) lo/36 (28%) 6/23 (26%)

e/12

4/18

III 41~60%

of leukemias with the following mean percentages of high H-2-bearing thymocytesc

Thymocytes

13 (10-20)

on Leukemic

1

a Female C57BL/6 mice, 6 weeks old, received an intrathymic injection of RLV (0.02 ml). Mice inoculated with D-RLV body irradiation. One group of mice was exposed to 4 weekly doses of 170 R whole body irradiation. b Index expressed as [(percentage of dead cells in antiserum) - (percentage of dead cells in normal serum)/(percentage x 100. c Anti H-2h serum used for C57BL/6 mice and anti H-2b for AKR/J mice. d Number of mice from total leukemic mice tested.

100 100 220

240 120

Leukemia

70-100 O-20

method” Latency (days)

induction

Incidence (%)

Leukemia

Surface Antigens

TABLE

$ $ z !3 2:

3

c3 r

!2

z 1: I/,

z 5

0

2 z

2 c [I,

Id w

360

CHAZAN

AND

HARAN-GHERA

TABLE Comparative

2

Study of High H-26 Alloantigen Expressed by Cytotoxic Indirect Immunofluorescent Test

Source of tested cells

High H-2b thymocytes Cytotoxic test

(‘Q

Surface immunofluorescence

Test versus

Background test, Cells bearing IgG (%I Direct immunofluorescence

Normal thymus, 2 months

Thymoma,

D-RLV

Thymoma,

D-RLV

C57BL/6,

=

400 R

10 18 2 12 13

12 16 6 12 15

0 0 0 1 0

78 80 6.5 70

80 85 75 80

2 4 1 1

75 69 86 74 8.5

8.5 77 90 85 92

1 1 4 0 1

o The level of high H-2” thymocytes was determined by exposing the same batch of leukemic or normal thymocytes from individual mice to the cytotoxic assay and indirect immunofluorescent test using the same H-2b antiserum.

within the four different categories (including 2.5% of leukemias with the normal thymus pattern) was observed among the A-RLV-induced leukemias. The localization and semi-quantitation of isoantigens on the cell surface of leukemic thymocytes versus normal thymocytes were demonstrated using the membrane immunofluorescent reaction (7). Indirect staining of living leukemic thymocytes in comparison to living normal thymocytes with highly labeled rabbit IgG anti-mouse IgG followed by alcohol fixation indicated the presence of confluent patches of fluorescence on the majority of leukemic thymocytes, whereas only a few tiny spots (two to four) were observed on the majority of normal thymocytes. The resemblance between the results demonstrating the incidence of high H-2b-bearing thymocytes (normal and leukemic cells) using the cytotoxic test versus the indirect immunofluorescent method is expressed in Table 2. A survey of the antigenic properties of the cell surface of mouse thymocytes has indicated that, whereas the major thymus subpopulation possesses high levels of 13antigen, the minor population bears low levels of 19and high levels of H-2. It seemed therefore of interest to analyze the characteristics of leukemic cells concerning the level of 8. The modified cytotoxic assay described by Shortman and Jackson (8) that provides selective killing of high 0 cells (by lowering the anti-8 level and the complement level and by reducing the incubation period) was used. The anti-6 antiserum dilution used in the present study was 1: 26, and the mixture

82 It 3 86 f 2 90 f 3

6 10 6

Cells

1.5 f 2 81 f 1 21 *4

3.5 f4 20 f 3

83 f 5 14 f 3 16 f 7

High 0 cells (1: 26 serum dilution) (mean %>

H-2b H-2k H-2k

H-2b H-2b

H-2b H-2b H-2b

W-2)

2 3

3 2 4

88 f 2 12 f2 85 f 3

93 f 87 f

16 f 90 f 67 f

(mean %I

High H-2 cells (1: 16 serum dilution)

0 Six- to eight-week-old female C57BL/6 mice received an intrathymic injection (0.02 ml) of D-RLV and 2 days later were exposed to 400 R whole body radiation or A-RLV alone. The thymomas occurred after an 80-100 day latent period. The spontaneous AKR/J thymomas were collected from 14-month-old mice. The normal thymus was taken from 2-month-old mice. The validity of the assay was confirmed by including a group of 2-month-old C57BL/6 mice treated with hydrocortisone acetate, each mice receiving 2.5 mg ip, and its thymus subjected to analysis 3 days later. The assay procedure is described in the text.

92 f 3 86 zk 2

14 6

89 f 4 78 f 3 81 h.5

0 cells (1: 16 serum dilution) (mean %I

D-RLV = 400 R A-RLV C57BL/6 + 2.5 mg of hydrocortisone acetate ip Normal AKR/J Thymoma, AKR/J spontaneous

Mice tested (No.1

12 8 6

of host, donor’

Assay for High &Bearing

3

Normal C57BL/6 Thymoma, D-RLV 2 400 R Thymoma, A-RLV 30 Days after virus inoculation

Treatment thymocyte

Cytotoxic

TABLE

362

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AND

15

HARAN-GHERA

20

25

30

35

Days post lost x-my

FIG. 1. Mean incidence of high H-2b alloantigen-bearing thymocytes (using cytotoxic assay) m 6-8-week-old C57BL/6 female mice, following each fractionated radiation exposure. The analysis was started 5 days following the first, second, third, or fourth exposure to X-rays. Each radiation dose consisted of 170 R whole body irradiation, and the interval between the repeated exposures was 7 or 14 days. Each point includes analysis of 12-14 individual mice. Thymus weight curves following X-ray exposure are charted in the inset.

of thymocytes-antiserum was incubated for 15 min at 4°C. One-tenth milliliter of 1 :lO-diluted complement was added, and the mixture was incubated for only 10 min at 37°C then immediately cooled in an ice bath. The cells killed were considered high d-bearing thymocytes. Spontaneous AKR leukemias, radiation leukemia, and Z-month-old normal thymuses were tested. The results obtained are summarized in Table 3. The percentage of thymocytes bearing high levels of 0 was much reduced, when compared to their distribution in the normal thymus, both in spontaneous and induced thymomas ; these findings were in accordance with the increased incidence of thymocytes bearing high levels of H-Z alloantigen among these leukemic thymocytes (Table 3). The efect of Leukelnogenic

Agents on Thymus Subpopulation

Patterns

Since the thymus is the target organ for the development of T leukemias found to bear the antigenic characteristics of the minor thymus subpopulation, it seemed plausible to assume that different leukemogenic agents could, perhaps, change the normal pattern of thymus subpopulations and, thereby, provide the susceptible thymocytes for overt leukemia development. Tests were performed to find out whether administration of the radiation leukemia virus or exposeure to fractionated whole body irradiation, both agents inducing a high incidence of lymphatic T leukemias in the C57BL/6 strain of mice, could change the thymus lymphoid population pattern transtiently for several weeks after leukemogenic treatment, before leukemic cells populated the thymus. The present tests, using the cytotoxic assay, were concerned with the incidence of thymocytes bearing high levels of H-2 alloantigen within 40-50 days following different leukemogenic treatments,

THYMUS

SUBPOPULATIONS

IN

T LECJKEMOGENESIS

363

Radiation Eflects Female 6-S-week-old C57BL/6 mice were exposed to one, two, three, or four weekly doses of 170 R whole body irradiation, four doses being the optimal conditions for inducing a high leukemia incidence of 70-100% at an average latent period of about 200 days (9). Since the length of the interval between the repeated exposures was shown to affect leukemia incidence (lo), one group of mice was exposed to four doses of 170 R whole body radiation at 1Cday intervals between the repeated exposures (this treatment yielded a 35% incidence in the present experiment versus 75% at the weekly interval exposures). The results, expressed as mean percentages of thymocytes baring high levels of H-2 within 35 days following the last radiation treatment, in the different groups are indicated in Fig. 1. Radiation injury to the thymus was reflected in drastic reduction in thymus weight (in the present experiment from 74 mg normal weight to about 25 mg), due to elimination of the radiation-sensitive thymocytes, and the cells surviving radiation injury showed high levels of H-2” alloantigen, as expected. The effective leukemia induction system of four weekly exposures to 170 R did indeed change, for several weeks, the pattern of the normal thymus population. An abundance of high H-2 thymus-derived lymphocytes were present in the thymus (40-70%) in spite of the fact that the thymus had already regenerated and almost regained its initial normal weight (Fig. 1, upper right insert). It is interesting to stress the reduced incidence of high H-2”-bearing thymocytes (within the range of 15-25s) obtained when four doses of 170 R were given at 14-day intervals (a treatment yielding a low leukemia incidence), though changes in the thymus weight curve were similar in both groups. Three weekly exposures induced an increase in the high H-2 population beyond the normal level only within the first 14 days after radiation treatment, while one or two weekly exposures had a very slight effect on the thymus pattern besides the initial radiation injury. To exclude the possibility that the change in the thymus subpopulation pattern is the result of neoplastic cell proliferation in the thymus, we tested the presence of leukemic cells in the thymus of mice 14 or 28 days following the last X-ray treatment (four weekly expousres) by taking from each thymus subjected to H-2 analysis 3 x lo7 cells and injecting them intravenously into syngeneic mice exposed l-2 hr previously to 400 R whole body irradiation, thereby providing better conditions for leukemic cell proliferation (transfer of cells from one donor to one recipient). The leukemia incidence obtained was 8% (l/12) after 14 days and 20% (3/14) after 2s days, the latent period in both groups being 150-1SO days. Although most of the lymphatic leukemias induced by irradiation in the C57BL strain of mice originated in the thymus, radiation exposure to the thymic area did not cause leukemia development (11) . Prevention of radiation leukemogenesis was afforded by shielding the spleen or bone marrow during exposure of the rest of the body to X-rays or by injecting isologous bone marrow or spleen cells into the irradiated hosts shortly after termination of the radiation treatment ( 11). Would these means that effectively prevent leukemia development, in spite of the radiation exposure, change the thymus subpopulation patterns observed after treatment with the highly effective induction system? To clarify this problem, four groups of mice (each consistin g of SO mice) were exposed to four weekly doses of 170 R whole body irradiation. The first group served as a control; shielding of bone marrow was introduced at the last exposure of the second group ; the third

364

CHAZAN

AND

IO

15

HARAN-GHERA

pf30‘6 -2 70N + 60+n E 508 5 402 30;:-+.. I

5

20

25

30

35

Days post last x-ray

FIG. 2. The effect of preventive methods in radiation leukemogenesis on the incidence of high H-2b-bearing thymocytes. Female C57BL/6 mice (68 weeks old) were exposed to four weekly doses of 170 R; 2 X 10’ isologous bone marrow cells or 5 X 10’ spleen cells (from 8week-old female mice) were injected intravenously within 1-2 hr after the last radiation exposure. Bone marrow shielding was performed by using a lead shield on the hind legs during the last exposure. Each point consists of data from 10 mice.

group received an intravenous injection of 2 X lo7 isologous bone marrow cells within l-2 hr after the last radiation treatment, and the fourth group was similarly treated with spleen cells (5 X 10’). These additional treatments that prevented leukemia development (85% in the control versus 45% with spleen cell treatment, 30% with bone marrow injection, and 15% with bone marrow shielding), reduced drastically the radiation-induced elevated high H-2 population pattern as seen in Fig. 2. All the “preventive” treatments in the present study induced an increase in thymus weight beyond the normal control curve, thereby indicating the actual effect of the injected cells on the thymus. Bone marrow shielding during the last exposure reduced the level of this minor population below the normal level already 7 days after treatment, and injection of bone marrow cells had a similar effect. The injection of spleen cells was less effective than bone marrow treatment, maintaining a level of 45-25s incidence of thymocytes bearing a high level of H-2” for about 3 weeks after last radiation treatment, compared to 70-40s for 28 days without any further treatment. Radiation Leukemia Vims Effects Cell surface antigens. Tests were performed to find out whether administration of the two variants of the radiation leukemia virus (that have a different leukemia induction capacity when introduced without any further co-leukemogenic treatment) could change the normal thymus pattern. The presence of high levels of H-2 alloantigen on thytnocytes within 50 days following virus injection intraperitoneally (ip) or intrathymically (it) was determined using the cytotoxic test (trypan blue dye exclusion test). C57BL/6 mice, 6-8 weeks old, received a single injection of D-RLV or A-RLV ip (0.5 ml) or it (0.02 ml), the same virus preparation being used for the two routes of administration. Both virus variants when injected

THYMUS

SUBPOPULATIONS

IN

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36.5

FIG. 3. Mean incidence of high H-2’ alloantigen-bearing thymocytes in C57BL/6 mice following intraperitoneal (i.p., 0.5 ml) or intrathymic (it, 0.02 ml) injection of the radiation leukemia virus variants D-RLV or A-RLV. Thymus weight curve following virus injection in upper right inset. Each point consists of data from 10 mice.

intrathymically induced a marked elevated level of 65-95s of thymocytes bearing high H-2 alloantigen for at least 30 days after virus inoculation. The intraperitoneal virus inoculation, a route less efficient for leukemia induction than intrathymic inoculation, was less effective and induced a reduced elevation (up to 50% for 30 days, with A-RLV reaching normal level at 40 days; 60% for about 20 days with D-RLV, and beyond 30 days below the normal level of thymus high H-2 subpopulation) (Fig. 3). It should be stressed that thymus weights within the period of l&20 days following intrathymic virus injection ranged between 60 and 70 mg, namely a “normal” thymus weight. A-RLV when injected intrathymically into Fr hybirds (Balb/c X C57BL/6) lost its high leukemogenic induction capacity, leukemia incidence being O-20% and increased to 70-1000/o when such inoculated mice were further exposed to 400 R whole body irradiation, namely, acquired the characteristics of D-RLV, and induced a shorter transient change in thymus pattern: 70% within the first 20 days and back to normal levels within 4 weeks after virus injection, similar to the D-RLV thymic effect. The leukemia incidence induced by D-RLV is low (O-20%) but can be increased to SO-100% by further exposure of the inoculated mice to X rays (200-400 R) (2). A comparative analysis of thymus patterns induced by methods that yield a high leukemia incidence, including the suitable control groups, was carried out. Male C57BL/6 mice, 6-8 weeks old, received a single intrathymic injection (0.02 ml) of D-RLV, A-RLV, or PBS (the solution used for RLV preparation) and some of the D-RLV- or PBS-injected mice were exposed to 400 R whole body irradiation 3 days after virus inoculation. The relative presence of 6 antigen and high levels of H-2 alloantigen on thymocytes 30 days following the leukemogenic treatment or on virus-induced thymomas was first evaluated by calculating thymocyte lysis in relation to antiserum dilutions (Fig. 4). The titration curve for 8 levels was similar for normal thymus and

366

CHAZAN

AND

HARAN-GHERA

14 Fmol

Dilulmn

of AntI 6 And

I 16 Anti

164

1256

1'1024

H-Zb

FIG. 4. The relative presence of 0 antigen and levels of H-2” alloantigens on C57BL/G normal, preleukemic, or leukemic thymocytes in relation to antisera dilutions. Each point is calculated as the mean of values from five mice tested.

thymomas, whereas the anti-H-2” serum killed the majority of thymoma cells and thymocytes following virus inoculation at similar dilutions in contrast to normal thymocytes that were less sensitive to antiserum lysis. Further analysis was performed in relation to high and low leukemia induction methods and the results obtained are summarized in Fig. 5. Treatments ultimately yielding a high leukemia incidence induced an abundance of high H-2-bearing thymocytes (60-75 % ) within the latent period, until overt leukemia occurred, whereas D-RLV alone in the present test triggered the occurrence of this subpopulation .transiently (a level of 927 o within 20-35 days following virus injection, thymus weight being about 60 mg, and a decrease to 25-35 % between 45-60 days). It was interesting to observe the effect of 400 R alone on thymus subpopulations; the minor high H-2 population remained below the normal level for many weeks, although thymus weight returned to normal level within 25-30 days following radiation exposure. Other characteristics of the minor high H-2 thymus subpopulation include low levels of 8, relative resistance to cortisone, and the capacity to cause a graft versus host (GVH) response. Are these characteristics also shared by thymocytes bearing high levels of H-2 alloantigen following leukemogenic treatment? The results concerning cytotoxic assays for low 0 and high 6’ cells 30 days after treatment with D-RLV plus radiation or A-RLV are given in Table 3. The majority of these thymocytes (85-95s) had high levels of H-2b and a reduced incidence of high 0 cells. The mean percentage of high 6’ cells was 30-40s in D-RLV + 400 Rtreated mice, and 15-25s in the A-RLV treated mice, versus S&90% among normal thymocytes, figures similar to those obtained in thymomas induced by these agents. Sensitivity to corticoids. The resistance of thymocytes bearing high levels of H-2 to cortisone (due to A-RLV treatment) was demonstrated in the following experiment : Female C57BL/6 mice, 6 weeks old, received an intrathymic injection of A-RLV or PBS (0.02 ml), and 30 days later half of these treated mice received a single intraperitoneal injection of 1 mg. Hydrocortisone acetate and, 24 hr thereafter, thymus weights and the level of the high H-2”-bearing thymocytes

THYMUS

SUl3l’OPULATIONS

IO

20

30

IN

40

Days following

367

T LEUKEMOGENESIS

50

60

treatment

FIG. 5. Incidence of high H-2b-bearing thymocytes following leukemogenic treatment yielding high leukemia incidence (A-RLV or D-RLV followed by 400 R) versus low leukemia incidence (D-RLV or 400 R). Each point consists of data from 12-14 individual mice. Thymus regeneration weight curve in the inset.

(using the cytotoxic test) were recorded. The results obtained are summarized in Table 4. The virus itself had a certain thymolytic effect, and further mean thymus weight reduction due to hydrocortisone treatment was 25 versus 6S% obtained in normal mice treated similarly. The incidence of thymocytes bearing high levels of H-2 was markedly increased following hydroctorisone injection into normal young mice; the A-RLV treatment itself increased the level of this subpopulation to S5% so that the hydrocortisone effect was not effective at this point. Graft versus host reactivity. Thymocytes bearing low levels of 0 antigen and high levels of H-2 are considered to be the immunocompetent cells able to cause TABLE Cortisone

Effect on Virus-Induced

Treatment of thymus donors

Normal Normal

control + hydrocortisone

A-RLV

Thymus weight (w)

4 High H-2 Thymus

Subpopulation

High H-2b thymocytcs (1: 8 dilution) (%I

76 i 2 24 * 4

;I 1;

50 * 3

85 f

3

36 3~ 1

90 f

3

}

Thymic weight reduction (%)

68

}

35

28 A-RLV

+ hydrocortisone

a C57BL/6 mice (6 weeks old) received an intrathymic injection of A-RLV or PBS (0.02 ml) and 30 days later part of them received a single injection of hydrocortisone acetate, 1 mg ip, and 24 hr later thymus weight and H-2b thymocyte levels were tested (each value is the mean for 10 thymuses).

368

CHAZAN

MO cells

AND

2X10’ cells

3x107 cells

HARAN-GHERA

4x10’ cells

5x10’ cells

IXO’ cells

FIG. 6. The GVH reactivity of thymocytes 20 days following intrathymic injection of A-RLV into 6-week-old C57BL/6 mice. (Balb/c X C57BL/6) F, lo-day-old mice (8-10 mice per group), received an intraperitoneal injection of thymocytes or spleen cells from CS7BL/6 donors, and 10 days later the spleen indexes were calculated.

a GVH response. We therefore tested whether the increased high H-2 population within the thymus following A-RLV inoculation could be correlated with an increased capacity to induce a GVH response. The GVH reactivity was determined by Simonsen’s method (12). T wo-month-old C57BL/6 mice received an intrathymic injection of A-RLV or PBS (0.02 ml), and 20 days later these mice served as thymus cell donors. Thymus cells, 1-5 X 107 cells were injected intraperitoneally into lo-day-old (Balb/c x C57BL/6) Fr mice (S-10 mice per group), and 10 days later their spleen index was estimated ( lo7 spleen cells from 2-monthold C57BLJ6 mice were also injected into Fr mice serving as the positive control group). Thymocytes (5 X 10’ cells) from A-RLV-treated donors were able to evoke a GVH (mean spleen index 2.22 * 0.13 versus 1.71 + 0.13 with the same number of thymus cells from normal donors) response similar to lo7 spleen cells (2.28 + O.OS), and the different amounts of thymus cells from A-RLV donors indicated a higher response than normal thymocytes except the low cell dose of lo7 Cells (1.03 2 0.06 compared to 1.10 + 0.07 with normal thymus cells) (Fig. 6). Thus, enrichment of thymocytes capable of inducing a GVH response resulted from intrathymic virus inoculation (effects similar to those produced by hydrocortisone treatment). Transplantation bioassay. To exclude the possibility that alterations in thymus subpopulation patterns were due to neoplastic cell proliferation in this organ (to cells that could not be detected by histological examination), the following thymus cell transfer experiment was performed. Thymus cells from C57BL/6 mice treated with A-RLV, D-RLV, D-RLV plus X rays, or PBS were collected 15 and 30 days after the intrathymic injection and transferred into Fr (Balb/c X C57BL/6) adult mice within 1-3 hr following their exposure to 400 R whole body irradiation (thymus cells were transferred from one donor to one recipient iv). The use of hybrid mice in this test enabled the identification of the leukemic cell genotype whether of donor or recipient origin by transplanting the tumor cells developing in Ft hybrid mice into F1 and parental strains. The origin of leukemic thymocytes from the parental C57BL/6 donor strain would indicate the actual transfer of established leukemic cells among the transferred thymus cells, whereas leukemias of F1 host origin would indicate the presence of a leukemogenic noncellular agent among the transferred cells that caused neoplastic transformation and proliferation in the thymus or other lymphoid organs of the host. The results of these transfer

15 30

Fr + 400 R FI f 400 R

(73%)

13114 -

(92%)

12/12 (100%)

12/16 (75%)

D Donor mice: Female C57BL/6 mice, 8 weeks old, received intrathymic injection of A-RLV, D-RLV, were further exposed to 400 R whole body radiation. b Recipient hosts: Eight-week-old female (BaIb/‘c X C57BL/6)Fr mice were exposed to 400 R whole following RLV or PBS inoculation (transfer of thymus from one donor to one recipient) were injected kemias occurring in the Fr recipient mice were further transferred (10’ leukemic cells) to (Balb/c X analysis.

PBS

Fr + 400 R FI + 400 R

15

30

R

D-RLV

FI + 400 R Fr + 400 R

1 l/IS

Mice

9/11

lO/lO

9/10 s/10

9/10 4/14

Recipient

Leukemia

2/1t

-

t/t0 2/10

l/10 10/14

Donor

origin

mice body irradiation. Thymus cells collected 15 or 30 days intravenously l-3 hr after exposure to X-rays. The leuC57BL/6)Fr and parental C57BL/6_mice for genotype

or PBS (0.02 ml), and part of the D-RLV-injected

-

112

106

110 113

140 75

(70%)

12/18 (66%) 14/20

incidence

C57BL/6

F1$-NOR

Leukemia

Cells from RLV-Treated

F, + 400 R

5

Latency (days)

b

15 30

1400

TABLE of Thymus

Incidence

Recipients

Transfer

D-RLV

Following

15 30

Time of transfer after treatment (days)

Induction

A-RLV

Donor treatment”

Leukemia

370

CHAZAN

AND

HARAN-GHERA

TABLE Mitotic

Index in Thymus

Days after treatment

6

Following

D-RLV

Injection”

Controls Number tested

7

10

14

12

D-RLV Mean mitotic indexb 2.2 (2.0-2.5)

Number tested

injected Mean mitotic index

10

8.6 (3-20)

12

7.2 (559.0)

(2.Z.O) a Two-month-old C57BL/6 mice received an intrathymic injection of D-RLV colchicine was injected ip 24 hr before thymus chromosome analysis. b Mitotic index = [(number of cells in mitosis)/5001 X 100.

or PBS (0.02 ml) ;

experiments are summarized in Table 5. The occurrence of leukemic cells in the thymus within 15-30 days following RLV treatment differed depending on the RLV variant tested. The leukemias developing after injection of thymus cells from D-RLV or D-RLV plus X rays at both time intervals were mostly of recipient origin probably due to a noncellular leukemogenic agent present among the transferred thymus cells (Table 5). Lack of demonstrable leukemic cells would therefore suggest that the change in thymus subpopulation patterns in D-RLV-treated mice was not due to leukemic thymocytes. Different results were obtained when thymus cells collected from A-RLV-treated donors were transferred. Thymocytes taken from A-RLV donors 15 days following virus inoculation when transferred in F1 recipients induced leukemias mostly of recipient origin (indicating virus transfer), whereas after 30 clays 70% of the induced leukemias were of donor origin, namely, the thymus was already populated with leukemic cells. Thus, the variable changes of thymus subpopulations were not necessarily related to the presence of leukemic cells. Mitotic index. The increase in the high H-2 thymus subpopulation following virus inoculation was reflected in a significant increase in thymic mitotic index. D-RLV or PBS was injected intrathymically, and the thymic mitotic index was tested 7 and 14 days following the intrathymic injection. The mice received an intraperitoneal injection of colchicine 24 hr before thymus removal. The ASG method described by Bukland et al. (13) f or identifying chromsomes by producing characteristic banding patterns on thymocyte metaphase chromosomes was used in the present analysis. As seen in Table 6 a significant increase in the mitotic index, induced by D-RLV injection intrathymically (the high H-2 level in the thymocytes tested being 60-SO%), was observed. Thyn~s grafts. The possible host effects on thymus subpopulation patterns was tested using thymus grafts under the kidney capsule of “normal” or “T”-depleted mice. Ault C57BL/6 female mice (6-S weeks old) were thymectomized and 10 days after thymus removal these mice were further exposed to 750 R whole body irradiation and within 1-3 hr later reconsitituted with lo7 syngeneic bone marrow cells (taken from S-week-old C57BL/6 mice) iv. A control group of intact mice was similarly treated. A newborn syngeneic thymus (within 24 hr of age) was grafted

TIIYMUS

SUBPOPULATIONS

IN

T LEUKEMOGENESIS

371

FIG. 7. The mean incidence of thymocytes bearing high levels of H-2 within isologous thymus grafts under the kidney capsule of “T”-depleted mice (obtained by adult thymectomy, lethal irradiation, and bone marrow reconstitution), or intact, irradiated, bone marrow-reconstituted mice, following D-RLV inoculation into 30-day-old intrarenal thymus grafts. Each point represents the mean of values from 10 thymus grafts.

under the kidney capsule 21 clays following radiation-bone marrow treatment, D-RLV or PBS was injected directly into the graft when it was 30 clays old (0.02 ml, the graft weight being 40-55 mg), and at different time intervals thereafter the levels of graft thymocytes bearing 0 and high levels of H-2b were cletermined. Virus inoculation increased markedly the percentage of thymocytes bearing high levels of H-2 irrespective of the presence or relative absence of T cells in the host ; the percentage of B-bearing cells in the spleen of thymectomized irradiated bone marrow-treated hosts tested 4, 11, 25, and 34 clays after injection was l-2% compared to 21-287 0 in spleens of intact mice similarly treated at the same time intervals (Fig. 7). A similar increase in the mitotic index found in intact thymuses following virus inoculation was also confirmed in thymus grafts irrespective of host lymphoicl populations. The mean mitotic index in virus-inoculated grafts (10 mice) in “T”-depleted hosts (2 weeks after injection) was 6.9 -+ 2.2 versus 2.6 -+ 0.5% in the PBS-injected ocntrols and 7.5 t l.S% in virus-inoculated grafts residing in intact, irradiated, bone marrow-reconstituted mice as compared to 2.2 -+ 0.4% in the controls. AKR Thywms Subpopulations Inoculation of the leukemogenic virus RLV into C57BL/6 mice induced changes in thymus subpopulation patterns. Since infectious virus has been demonstrated in many tissues of the embryo or adult AKR mice in the preleukemic periods (14)) it seemed of interest to test the age-related thymus population in this strain of mice. The mean percentage of high H-2- and O-bearing thymocytes was evaluated in nonleukemic AKR thymuses taken from l-15-month-old mice ( 10-12 individual thymuses per group were tested; the upper half was removed for sectioning). The results obtained are charted in Fig. S. A significant spontaneous increase in

372

CHlZZAN

I I

I 3

AND

I 5

, 7

HARAX-GHER-4

I 9

I II

I 13

I 15

J

Age of mce (months) FIG. 8. Age-related mean incidence of high H-2k- and B-bearing thymocytes in AKR/J Each point consists of data from IO-12 individual mice.

mice.

the high H-2 thymus subpopulation was observed from 4-S months onwards, reaching a 45% incidence in S-month-old nonleukemic thymuses and above 70% in I-yr-old nonleukemic mice. A small but significant decrease in the percentage of &bearing cells was also observed from 10 months onwards. DISCUSSION The thymus is considered as the site for the development of leukemic cells and the organ chiefly involved in the neoplastic process. The sequence of changes occurring in a population of lymphoid cells undergoing neoplastic transformation seemed of special interest. In the present studies we have demonstrated that the majority of spontaneous AKR leukemias or T leukemias induced in C57BL mice by X rays or leukemogenic virus had cell surface antigenic characteristics of the minor thymus subpopulation, namely, low levels of 19antigen and high levels of H-2 alloantigen. The functional differences found by Barker and Waksal (15) among AKR lymphoid leukemic cells and the recent observations of Zatz (16) suggesting that the leukemogenic process in AKR mice selectively affected lymphocytes susceptible to PHA stimulation (though no generalized major deficit of T cells was indicated) might coincide with the variable incidence of thymocytes bearing high levels of H-2” described in the present study (Table 1). It could be assumed that the antigenic characteristics shared by leukemic thymocytes and the minor normal thymus subpopulation are due to the actual neoplastic transformation. Alternatively, leukemogenic agents could induce changes in thymus subpopulation patterns and thereby provide the susceptible population for thymocyte neoplastic transformation and/or leukemic cell proliferation. The present studies have indeed demonstrated that the different leukemogenic agents induced a transient or permanent chang in thymus population (identified by its surface markers and functional properties). The possibility that the changes in thymus subpopulations within several weeks following leukemogenic treatment

THYPIUS

SUBI’OPULATIOKS

Iru‘

T LEUKEMOCENESIS

373

coincided with the occurrence of leukemic cells in the thymus (cells that could not be detected by histological examination) was excluded by transplantation bioassays, using syngeneic or F 1 hybrid recipients. The method (17) used for identification of preleukemic cells in the thymus, including genotype analysis of the leukemias developing in F1 hybrids within a long latent period, indicated in most tests that the “preleukemic” thymus did not contain transplantable neoplastic lymphoid cells (since most of the developing leukemias were of host origin) following intrathymic inoculation of D-RLV alone or D-RLV plus radiation treatment. The one exception, following A-RLV treatment (the majority of leukemias being of donor orgin when thymocytes were transplanted 30 days after A-RLV intrathymic inoculation), further indicated that changes in the thymus population occurred irrespective of the presence or absence of preleukemic cells in the thymus. Metcalf’s findings concerning morphologic abnormalities in the preleukemic AKR thymus involving depletion of small lymphocytes from the cortex and the development of lymphoid follicles in the medulla (18) coincide with the induced changes in thymus subpopulation patterns demonstrated in the present studies. The correlation obtained between the availability of the transient elevated high H-2 thymus population, for several weeks, and leukemia incidence following fractionated radiation treatment was quite remarkable. Bone marrow shielding during the last radiation exposure or bone marrow administration within l-2 hr after the last radiation treatment reduced drastically the radiation-induced elevated high H-2 population pattern. The lack of this thymus subpopulation could, perhaps, be considered as a factor in preventing the proliferation of “preleukemic” cells found in the bone marrow of such treated mice (19). The fact that one single marrow shielding or administration of bone marrow cells during or after the last radiation exposure is as effective in preventing leukemia development as repeated “preventive” treatments after each radiation exposure coincides with the present findings that the susceptible high H-2 thymus subpopulation was present in abundance only after the last radiation exposure. The effect of the length of time intervals between repeated exposures on lukmia incidance was also reflected in the thymus subpopulation pattern. A marked reduction in the availability of the high H-2 population was observed when the fractionated exposure was done at a 14-day interval instead of a 7-day interval. Since there seems to be a correlation between the radiation treatment yielding a high leukemia incidence and a transient thymus pattern consisting mainly of thymocytes bearing high levels of H-2 alloantigens, it might be proposed that a certain level of this population is a prerequisite for further overt leukemia development. Similar changes in thymus population patterns, although thymus weight was similar to the normal control weight, were obtained following inoculation of the radiation leukemia virus variants, irrespective of their route of administration. Within several weeks following virus injection, there was a relative enrichment of the minor subpopulation of thymus cells bearing low levels of 0 and high levels of H-2 alloantigens. This population accounts for most of the GVH and PHA reactivity in the thymus (20, 21) . Indeed, thymocytes from virus-inoculated mice were partially resistant to the cortisone effect (22) and were capable of inducing a GVH response. The high susceptibility of newborn mice injected with D-RLV to develop leukemia (11) might, perhaps, be related with the findings of Droege et al. (23)

374

CHAZAN

AND

HAKAN-GHERA

(II and 111) respon~110 showed that the newborn thymus contains the cell types sible for GVH whereas during the first week of life another cell type (I) appears and persists into adult life. It was interesting to observe the effect of 400 R alone on thymus subpopulations (Fig. 5). The transient increase in the minor high H-2 population, following X ray elimination of the radiation-sensitive thymocytes, lasted only several days, and from about 10 days onwards remained below the normal level for many weeks, though thymus weight regained its normal level within 25-30 days following radiation exposure. A similar radiation effect on this decreased high H-2 thymocyte population was observed in mice exposed to fractionated irradiation plus reconstitution by bone marrow or spleen cells (Fig. 2). It has been shown that the active thymus helper cells are contained in the thymus hydrocortisone-resistant cell population (24). It could, perhaps, be proposed that the occurrence of T suppressor cells following radiation may actually be affected by the reduced incidence of the thymus minor cortisone-resistant subpopulation that was demonstrated to have “helper” activity in humoral immune reactivity (20). The follow-up of changes in the population of thymus grafts in T-depleted hosts, being similar to those obtained in situ in the thymus of intact virus-inoculated hosts, and the similar increase in the mitotic index obtained in the graft and the intact thymus might suggest that an endogenous “factor” within the thymus is responsible for the observed changes in thymus population patterns. These findings are in agreement with Metcalf’s concept of thymus “autonomy” in relation to thymus development and involution regardless of the age of the host (25). Administration of the leukemogenic virus in C57BL mice induced a change in the thymus subpopulation, thereby providing an abundance of the high H-Zbearing thymocytes. Since we proposed that this population might be a prerequisite for leukemia development, it seemed of interest to study the thymus population pattern in the AKR strain of mice having an intrinsic leukemogenic virus source responsible for the development of spontaneous leukemia. We did indeed find a significant age-related spontaneous increase in the high H-2 thymus subpopulation from 5 months onwards and a gradual decrease in e-bearing thymocytes from 7 months. The increased PHA responsiveness, associated with the minor low 8 subpopulation, found in preleukemic AKR thymuses (26, 27), the decline in the T population of AKR mice predisposing onset of leukemia, and the decrease in 0 antigen sites (25, 29) coincide with the spontaneous age-related increase in the incidence of thymocytes bearing high levels of H-2 described in the present studies. The timing of the spontaneous changes in the AKR thymus pattern coincides with the gross proliferation of the disease in the thymus. Nagaya (30), studying the significance of the latent period in spontaneous leukemia development in the AKR strain by using thymus grafts in relation to age variations, has indicated that aging of the thymus was necessary for leukemia development. The observed age-related changes in the AKR thymus subpopulations in situ could affect the long latent period needed for overt spontaneous leukemia development in this strain. It should be stressed that the spontaneous age-related changes in thymus population characteristics were found to be specific for the high leukemia AKR strain, since similar studies in the C57BL strain (30, 31), as well as in C3H and Balb/c mice (unpublished observations) indicated a consistent low level

THYMUS

SUBPOPULATIONS

IN

T LEUKEMOGENESIS

(S-2070) of thymocytes bearing high levels of H-2 throughout tested were 30 months old).

375 life (oldest mice

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32.

Haran-Ghera, N., Znt. J. Cancer 1, 81, 1966. Haran-Ghera, N., Zsr. J. Med. Sci. 7, 17, 1971. Haran-Ghera, N., and Weiss, D. W., 1. Nat. Cancer Zmt. 50, 229, 1973. Haran-Ghera, N., Ben-Yaakov, M., Chazan, R., and Peled, A., In “Comparative Leukemia Research,” pp. 133. Karger, Basel, 1975. Shortman, K., Cerottini, J. C., and Brunner, K. T., Eur. J. Zntmunol. 2, 313, 1972. Winn, H. J., Ann. N.Y. Acad. Sci. 101, 23, 1962. 13, 395, 1967 Cerottini, J. C., and Brunner, K. T., Immzmology Shortman, K., and Jackson, H., Cell. Immunol. 12, 230, 1974. Kaplan, H. S., J. Nut. Cancer Inst. Monogr. 14, 207. 1969. Kaplan, H. S., and Brown, M. B., J. Nat. Cancer Iwt. 13, 185, 1952. Kaplan, H. S., Ser. Waematol. VIII, 2, 1974. Simonsen, M., Engelbreth-Holm, J., and Jensen, E., Ann. N.Y. Acad. Sci. 73, 834, 1958. Buckland, R. A., Evans, H. J., and Summer, A. T., Exfi. Cell Res. 69, 231, 1971. Rowe, W. P., and Pincus, T., J. Exfi. Med. 135, 429, 1972. Barker, A. D., and Waksal, S. D., Cell. Immwzol. 12, 140. 1974. Zatz, M. A., J. Zm~nz~nal. 115, 1168, 1975 Haran-Ghera, N., Nature (Loxdon) 246, 84, 1973. Metcalf, D., J. Naf. Cancer Inst. 37, 425, 1966. Haran-Ghcra, N., In “Biology of Radiation Carcinogenesis,” Raven Press, New York, in press. Dyminski, J. W., and Smith, R. T., Ser. EIlacmatol VII, 4, 524, 1974. Shortman, K., Bochman, H. V., Lipp, J., and Hopper, K., Transplant. Rev. 25, 161, 1975. Blomgren, H., and Anderson, B., Cell. Immzmol. 1, 545, 1971. Droege, W., Zucker, R., and Janker, U., Cell. Immunol. 12, 173, 1974. Cohen, J. J., and Claman, H. N., J. Exp. Med. 133, 1026, 1971. Metcalf, D., In “The Thymus, Recent Results in Cancer Research,” Vol. 5. SpringerVet-lag, New York, 1966. Nagaya, H., J. Immzmol. 111, 1052, 1973. Zatz, M. M., Goldstein, A. L., and White, A., J. Immwzol. 111, 1514, 1973. Barker, A. D., Rhicns, M. S., and Pierre, L. R.. Cell. Immunol. 7, 85, 1973. Filppi, Y. A., Rheins, M. S., and Pierre, L. R., Immz~nology 28, 659, 1975. Nagaya, H., J. Immztnol. 14, 1048, 1973. Haran-Ghera, N., Chazan, R., and Ben-Yaakov, M., In “Fifth International Conference on Lymphatic Tissue and Germinal Centers in Immune Reactions,” Plenum Press, New York, in press. Delima, M. G., and Walford, R. L., Proc. SOL. Exp. Biol. Med. 149, 562, 1975.