J Cancer Res Clin Oncol (1981) I00:149-165 J o u ~ l of

~nCancer Research Clinical Oncology �9 Springer-Verlag 1981

T- and B-Cell Determination in Various Lymphoid Tissues of Mice During N-Nitrosobutylurea (NBU) Leukemogenesis*

M. Kraus and G. R.F. Krueger

Immunopathology Laboratories, Institute of Pathology, University of Cologne, D-5000 Cologne 41, Federal Republic of Germany

T- und B-Zellbestimmung in verschiedenen lymphatisehen Organen der Maus w~ihrend der Entstehung N-Nitrosobutyl-Harnstoff-induzierter Lymphome

Summary. T- and B-cell distribution in thymus, spleen, and lymph nodes has been investigated during chemical lymphomagenesis. N-nitrosobutylurea (0.04 g/100 ml) in drinking water was administered to 35-day-old male C57BL mice for a period of 60 days. As of week 11, 21 of 22 Nbu mice developed thy- mic lymphomas of lymphoblastic cell type which showed a general leukemic spread to spleen, lymph nodes, and several extralymphatic organs at week 16. Immunocytologic studies revealed the T-cell origin of these thymic type lym- phomas. Alteration of the T-cell system during latency period was represented by reduced thymic weight, cell numbers, as well as absolute and relative T-cell reduction. Absolute T-cell reduction was also observed in the spleen, although the reduction of splenic lymphoid cells was mainly caused by a decrease of B cells. In lymph node subnormal variations of theta-positive cells took place, whereas B-cell distribution was not altered. An impaired T- and B-cell function was suspected from preleukemic changes of lymphoid subpopulations. The re- sults are compared with other routine lymphomas and discussed with regard to a functional hypothesis of lymphomagenesis.

Key words: Chemical leukemogenesis - T- and B-lymphocytes

Zusammenfassung. In der Latenzzeit eines chemisch induzierten Lymphoms wurde die T- und B-Lymphozytenverteilung im Thymus, der Milz und im Lymphknoten untersucht. N-Nitrosobutyl-Harnstoff wurde in einer Dosie- rung von 0,04 g/100 ml an 35 Tage alte mfinnliehe C57BL-Mfiuse 60 Tage lang

* Supported by the Minister for Research and Science, State Government of North Rhine-West- phalia

0171-5216/81/0100/0149/$3.40

150 M. Kraus and G. R. F. Krueger

im Trinkwasser verabreicht. Ab der 11. Woche nach Beginn der Nbu-Verabrei- chung traten bei 21 yon 22 Nbu-M/iusen lymphoblastische Thymuslymphome auf, die sich in der 16. Woche generalisiert in der Milz, im Lymphknoten und in verschiedenen extralymphatischen Organen ausbreiteten. Die immunzytolo- gischen Untersuchungen liegen eine Einordnung dieser Lymphome als reine T- Zell-Lymphome des Thymus mit leuk/imischer Generalisierung zu. Die Ver/in- derungen des T-Zell-Systems w/ihrend der Latenzzeit waren durch ein reduzier- tes Organgewicht, Zellzahlen, sowie eine Verminderung absoluter und relativer T-Zellzahlen des Thymus gekennzeichnet. Ein absoluter T-Zellrfickgang fand auch in der Milz statt, obwohl die Verminderung lymphoider Zellzahlen dieses Organes vornehmlich durch den Riickgang der B-Zellzahlen verursacht wurde. Im Lymphknoten traten subnormale Schwankungen theta-positiver Zellen auf, wfihrend die B-Zellverteilung unver/indert war. Aus den Ver/inderungen der Lymphozytensubpopulationen in der Latenzzeit des Lymphomwachstums konnte eine Beeintrfichtigung der zellul/iren und humoralen Immunfunktion geschlossen werden. Die Ergebnisse wurden mit murinen Lymphomen anderer Genese verglichen und hinsichtlich einer funktionellen Theorie der Lymphom- entwicklung er6rtert.

Schliisselwiirter: Chemische Leukfimogenese - T- und B-Lymphozyten

Introduction

Leukemias and lymphomas in man and mice can be classified according to immu- nologic surface properties either as T-, B-, or null-cell neoplasms (Krueger 1979; Shevach et al. 1972, 1973), which may allow conclusions as to the state of differ- entiation and function of the leukemic cells (Krueger 1979; Seligmann 1979). In man, immunologic surface markers add to the different classifications of non- Hodgkin lymphomas (Krueger 1979; Stein 1976). In mice, theta-alloantigen as one differentiation marker for T-cells (Raft 1969; Reif et al. 1964) and membrane im- munoglobulins of B-cells are well demonstrated (Kincade et al. 1971; Rabellino et al. 1971; Takahashi et al. 1971). Large amounts oftheta-antigen are present on mu- rine cortical thymocytes, while mature thymic lymphocytes and peripheral T-lym- phocytes possess less theta-antigen (Aoki et al. 1969). Fluorescein-isothiocyanate (FITC)-labeled antibodies of known specifities can be used to detect above mem- brane structures of lymphoid cells (Coons et al. 1955).

In the present experiments a chemical lymphoma model was chosen for immu- nologic investigations during the latent period of lymphoma development. The ob- jective was to determine lymphocyte subpopulations in thymus, spleen, and lymph nodes, to reveal immunocytologic changes which could reflect an altered immune status at preleukemic and leukemic stages, to identify the tumor cell population im- munocytologically, and to follow up the pathogenesis of lymphoma development. A crucial influence of immune function on malignant lymphoma development has already been described earlier (Krueger 1971, 1972; Zoltowaska 1975). N-nitroso- butylurea was administered for lymphoma induction according to an experimental model described earlier (Hiai et al. 1973). The results are compared with others of viral and radiation-induced leukemias of mice.

T- and B-Cell Determination in Various Lymphoid Tissues of Mice 151

Material and Methods Fifty male C57BL mice (Jackson Laboratories, Bar Harbor, ME, USA) were fed N-nitrosobutylurea (Nbu) for 60 days beginning at the age of 35 days. The dry substance of Nbu (Serva Biochemica Heidel- berg, FRG) was stored at -20 ~ For use, 0.04 g Nbu was dissolved in 100 ml destilled water and ad- ministered in drinking water ad libitum. Fifty control mice obtained normal tap water alone. The Nbu solutions were restored every morning and drinking bottles were shielded from light.

Immunocytologic investigations of T- and B-cell distribution in thymus, spleen, and lymph node were done 2, 4, 8, 11, 13, and 16 weeks after the beginning of Nbu-feeding.

Antibodies against theta-1.2 antigen were produced in AKR mice by immunization with homogen- ized thymus tissue of C3H mice who share H 2 speeificities with AKR mice but differ in their alleles for theta-antigen (Reif and Allen 1964). Immunoglobulins were concentrated by ammonium sulphon- ate solution, precipitation, and labeled with FITC according to Spendlove (1966). Protein concentra- tion was 18.7 mg/ml. The cytotoxie titer against C57bl thymocytes was 1:1,024. Specificity was con- trolled by absorption studies with liver or brain tissue.

B-lymphocytes were identified by their membrane immunoglobulin receptors using FITC-labeled antisera against mouse immunoglobulins IgG1, IgG2, IgA, or IgM (Meloy Laboratories, Inc. Biolog- ical Products Division, Springfield, VA, USA). Optimal dilutions for all FITC-labeled antisera includ- ing anti-theta-serum were determined before use.

Suspensions of 2.4 x 10 6 viable lymphoid cells in 0.2 ml HBSS were incubated with 0.08 ml of the appropriate dilution of each antiserum for 20 rain in the dark at room temperature. After two washings the percentage of fluorescent cells/mononuclear lymphoid cells in suspension was evaluated by use of a Zeiss fluorescence microscope (HBO 200 W/4 lamp, epifluorescenee, filter B63, Ug5, 47, mirror FI 450). Ringlike, crescent-shaped, capping, and fluorescent features with at least ten distinct spots on the membrane surface were considered as specific fluorescence. Dead cells showed an unspecific diffuse staining (Kraus 1980; Rabellino et al. 1971). In selected specimens the viability of incubated lymphoid cells was controlled by the Trypan blue exclusion test and ranged between 91% and 98%.

The organ weights of thymus and spleen are listed as relative organ weights calculated in mg/g body weight. Absolute numbers of lymphoid cells were determined in thymus and spleen by calculating total cell number per organ from counting the cells in a selective fragment and relating their number to the weight of the entire organ. Homogeneous cell distribution was confirmed by histological controls. Therefore, absolute T- and B-cell numbers could be estimated.

Statistical evaluation of cytologic findings in experimental and control mice was done by means of the Wilcoxon test for two samples (Documenta Geigy, Wissenschaftliche Tabellen; J. R. Geigy AG, Basel, 1969).

Results

Ten of 50 N b u mice died before investigations could be done. Four ty N b u mice were autopsied; the first thymic lymphomas were observed histologically at week 11 after the start of Nbu-feeding. F r o m 40 autopsied N b u mice 31 were investi- gated for T- and B-cell d is t r ibut ion in thymus, spleen, and lymph nodes.

Histological Findings

As of week 11, 21 of 22 N b u mice showed mal ignan t thymic lymphomas. One lym- phoma at week 11, five at week 13, and all lymphomas at weeks 15 and 16 were generalized involving lymphatic and extralymphatic tissues. Dur ing Nbu-feeding an a t rophy of the thymus became evident macroscopically, whereas a t rophy of spleen and lymph nodes was no t as prominent . The gross a t rophy of lymphat ic or- gans between week 2 and week 8 was also verified histologically. Especially in the thymus of weeks 2 and 4 the cortex was atrophic. The spleen showed a homologous a t rophy of all parts. In lymph nodes, cortex, paracortex, or whole lymphatic organ was mutua l ly atrophic. Regressive changes of the secondary lymphat ic organs were not un i fo rm in a sense that an a t rophy of certain thymus-dependent or thymus-

152 M. Kraus and G. R.F. Krueger

independent regions of spleen or lymph nodes would have indicated an isolated change of a single lymphocyte subpopulation. At week 11 of the experiment all six autopsied Nbu mice showed diffuse lymphoblastic proliferations within the thy- mus beneath atrophic regions. One thymic lymphoma was located in a single thy- mic lobe, whereas the second lobe was still atrophic. A further mouse from this week already showed a systemic lymphoma involving mediastinum, spleen, lymph nodes, liver, and lung. Six of seven Nbu mice of week 13 had thymic lymphomas. Five lymphomas were also disseminated either with a local infiltration of medias- tinal tissue or a systemic manifestation in other lymphoid or non-lymphoid organs. All mice of week 16 showed generalized malignant lymphomas, involving thymus, spleen, lymph nodes, kidney, liver, lung, Peyer's patches and surrounding intestinal mucosa, heart, testicle, and epididymis. With the exception of one mouse from week 13 all mice between weeks 11 and 16 showed malignant thymic lymphomas.

Organ Weights and Cell Numbers

During Nbu feeding until week 8 the relative organ weight of thymus was reduced as compared to control mice, most strikingly at weeks 2 and 4 with an absolute minimum at week 4. At this time, also histological atrophy was most prominent. After termination of Nbu-feeding the relative organ weight increased continuously exceeding the control level between weeks 8 and 11 until week 16, when it surpassed the thymus weight of normal mice by about eight times. Similarly, the numbers of thymic lymphoid cells was altered. From week 2 until week 8 thymic lymphocytes were reduced. Between weeks 8 and 11 a drastic rise of lymphoid cell numbers above the control level occurred. At week 16 a slight decline parallel to the control results took place (Table 1).

During Nbu administration the relative organ weight of spleen was only slightly reduced with the lowest average value at week 2. A transitory rise in average organ weight at week 11 was caused by an early lymphomatous generalization with splenic infiltration of a single mouse. Omitting this mouse, the average splenic weight was still slightly subnormal. At week 13 the spleen weight was slightly ele- vated. A significant rise of splenic organ weight in Nbu mice above those of the controls was first observed at week 16, when the spleens of all investigated mice

Ta~el

Weeks Thymus/body weight Thymus ce l l s Spleen/body weight Splenic lymphoid cells (mg/g) (10 6 ) (mg/g) (10 6 )

Control NBU Control NBU Control NBU Control NBU

2 3.2(4) a 1.2(6) s 76(4) 6(6)s 3.2(4) 2.1 (6)s 39(4) 11(6)s 4 2.5(4) 0.7 (6) s 63(4) 3(5)s 2 .9(4) 2.5(6(- 55(4) 14(5)s 8 2.2(4) 1.3(6)- 68(4) 9(5)s 2 .6(4) 2.3(6)- 46(4) 21 (5)s

11 2.2(4) 8.6(6)s 8 6 ( 4 ) 216(5)- 3 .4(4) 9.6(6)- 68(4) 120(5)- 13 2.2(4) 12.2(5)s 9 0 ( 4 ) 264(5)- 2 .5(4) 4.3(5)- 68(4) 65(5)- 16 1.8(4) 16.8 (5) s 58(4) 227 (5) s 2.9(4) 21.5 (5)s 65(4) 343 (5) s

" Mean value (number of mice investigated) s Statistically significant according to Wilcoxon test: 2e =< 0.05

T- and B-Ceil Determination in Various Lymphoid Tissues of Mice 153

were infiltrated by a generalized lymphoma. As in the thymus, the average numbers of splenic lymphoid cells resembled largely the changes of splenic organ weights. Lymphoid cell numbers, although continuously increasing, were low between weeks 2 and 8, more prominently than splenic weigths. Though lymphoma growth involved the spleen of one mouse at week 13, the average value of lymphoid cell numbers was slightly decreased at this time. Accompanying the organ weight in- crease was a progressive increase of lymphoid cell numbers up to 340 x 106 cells at week 16 when the spleen was infiltrated by lymphoma (Table 1). In essence, a gross staging of Nbu-induced malignant lymphoma development shows the fol- lowing: during Nbu administration between weeks 2 and 8 a thymic atrophy was leading. A progressive increase of lymphoblasts in the thymus consistent with ini- tiating lymphoma occurred between weeks 8 and 13. Finally, there was a lymphatic and extralymphatic leukemic spreading of lymphoma cells between weeks 13 and 16.

I m m u n o c y t o l o g i c F i n d i n g s

Whereas T-cells in the thymus of control mice were found in the range of 66-84%, T cells in Nbu mice decreased to 31% already 2 weeks after initiation of Nbu ad- ministration. At this time, the lowest T-call numbers of Nbu mice were seen in thy- mus. Compared to normal mice the T-cell number of Nbu mice was even lower at week 4. Subsequently, T-cell percentage increased slowly up to 39% until the end of Nbu administration. Afterward, a prominent rise to 64% at week 11 paralleled the increase of thymic weight and lymphocyte number. At this time lymphoma de- velopment was noted in thymus. At week 16 another distinct rise of relative theta- positive cell numbers occurred. In relation to control mice T-cell numbers were sig- nificantly reduced from week 2 until week 11 and elevated at week 16 (Table 2). Random samples at the time of relative T-cell depletion of thymus showed no in- crease of B-cell numbers.

T-cells in the spleen were elevated constantly between weeks 4 and 11 and sig- nificantly at week 8. From week 13 a progressive increase of relative T-cell numbers was noted with 52% rising to more than 80% at week 16. At these dates T-cell values of Nbu mice differed significantly from those of control mice (Table 2). B cells were reduced during the entire investigation period. This was statistically sig-

Table 2

Weeks % Theta-positive cells

Thymus Spleen Lymph node

Control NBU Control NBU Control NBU

2 66(4) 31 (6)s 36(4) 33(6)- 42(4) 23(6)s 4 84 (4) 35 (5) s 36 (4) 42 (5)- 44 (4) 39 (5)- 8 74(4) 39(5)s 32(4) 45(5)s 47(4) 31(5)

11 80 (4) 64 (5) s 31 (4) 44 ( 5 ) - 53 (4) 48 (5) - 13 77 (4) 64 ( 5 ) - 34 (4) 52 (5) s 56 (4) 28 (5) s 16 70 (4) 87 (5) s 32 (4) 82 (5) s 60 (4) 80 (5) s

154 M. Kraus and G. R. F. Krueger

nificant at weeks 4, 13, and 16. The most striking change was the reduction at week 16, when T cells increased drastically and all Nbu animals showed splenic lym- phoma. As of week 11 the difference compared to the B-cell distribution of control mice became larger. Relative null-cell numbers defined by the absence of T and B characteristics of the spleen were elevated insignificantly at weeks 2 and 4 and showed no obvious changes in the following period compared to control mice.

In lymph nodes subnormal variations of T-cell numbers were observed from week 2 until week 13. The null-cell values behaved inversely and were elevated at weeks 2, 8, and 13. B-cell distributions were not altered during this period. At week 16, when thymic lymphoma had extended to lymph nodes, T cells exceeded the con- trol value. Corresponding B cells decreased prominently, whereas null cells were not obviously altered (Table 3).

During Nbu-feeding absolute T-cell numbers in the thymus' were reduced sig- nificantly. Between weeks 8 and 11 a rise of T-cells from 3 x 106 up to 140 x 106 followed exceeding the control value twofold. The T-cell increase in the thymus af- ter the end of Nbu administration continued until week 16. Absolute T-cell num- bers in the spleen were decreased until week 8 in contrast to the relative T-cell dis- tribution. A significant rise of absolute T-cell numbers in spleen at first could be observed at week 16. Absolute B-cell values in the spleen were prominently de- creased during the entire period of investigation. The null-cell increase at week 11 was caused by a single lymphoma which had widely spread as compared to other thymic lymphomas at this time (Table 4).

Combined Histological and Cytologic Findings

The thymus of Nbu mice was atrophic until week 8. Comparing the average values of lymphocyte numbers and theta-positive lymphocytes in the thymus suggests that thymus atrophy was mainly caused by a reduction of T cells (Fig. 5). In the spleen, absolute T-cell numbers decreased less strikingly and null cells were not altered, whereas especially B cells showed a significant reduction at this time (Fig. 6). In the following a T-cell accumulation was noted in the thymus between weeks 8 and 11 represented by relative and absolute T-cell numbers (Figs. 2, 5). At this time, Nbu

Table 3

Weeks % Immunoglobulin-positive ceils % Null cells a

Spleen Lymph node Spleen Lymph node

Control NBU Control NBU Control NBU Control NBU

2 66(4) 56(6)- 50(4) 42(6)- - 2(4) b 11(6)- 8(4) 35 (6) s 4 66(4) 47 (5) s 44(4) 45(5) - 2(4) 11(5)- 13(4) 16(5)- 8 73 (4) 59 (5)- 42 (4) 36 (5)- - 5 (4) -- 4 (5)- 11 (4) 34 (5) s

11 56 (4) 37 (5) - 37 (4) 37 (5) - 13 (4) 20 (5 ) - 10 (4) 16 (5) - 13 58 (4) 35 (5) s 39 (4) 45 (5 ) - 7 (4) 9 (5 ) - 5 (4) 27 (5 ) - 16 53(4) 6(5)s 36(4) 10(5)s 15(4) 12(5)- 5(4) 10(5)

a Theta- and immunoglobulin-negative cells b Negative sign indicates contemporary presence of T- and B-cell properties

T- and B-Cell Determination in Various Lymphoid Tissues of Mice 155

20

1S

10

mg/g

T . I weeks

Fig. 1 Relative organ weights (mg/g body weight). Thymus weight: controls (o), Nbu mice (o); spleen weight: controls (~), Nbu mice (I). Black bar indicates the period of Nbu administration started at the age of 35 days

administration had been terminated and thymic lymphoma growth developed. A similar T-cell proliferation occurred in the spleen with a delay of 5 weeks except for one mouse. This T-lymphocyte rise was accompanied by a B-cell decrease; the latter were absolutely reduced during the entire investigation period (Figs. 3, 6). At week 16 a comparable T-cell rise and B-cell reduction was also observed in lymph nodes (Fig. 4). The time sequence of organ involvement by a T-cell proliferation

Table 4

Weeks 106 Theta-positive cells 106IG-positive cells 106 Null cells Spleen

Thymus Spleen Spleen

Control NBU Control NBU Control NBU Control NBU

2 51(4) 2(6)s 14(4) 4(6)s 26(4) 6(6)s -1(4) 1(6)- 4 53(4) l(5)s 20(4) 6(5)s 36(4) 7(5)s --1(4) 2(5)- 8 50(4) 3(5)s 15(4) 9(5)- 3 4 ( 4 ) 12(5)s -2(4) - 1(5)-

11 69(4) 139(5)- 21(4) 52(5)- 3 8 ( 4 ) 15(5)- 8(4) 55(5)- 13 69(4) 169(5)- 23(4) 33(5)- 3 9 ( 4 ) 17(5)s 5(4) 12(5)- 16 41 (4) 198 (5) s 21 (4) 281 (5) s 35 (4) 18 (5) s 9 (4) 39 (5)-

156 M. Kraus and G. R.F. Krueger

100

50

% THYMUS

v / weeks

2 4. 8 1;I 13 16 Fig. 2. Percental distribution of theta-positive thymus cells: controls (o), Nbu mice (e)

can be deduced from relative T-cell values of thymus, spleen, and lymph nodes which signify clearly the thymus as primary source of T-cell spread (Figs. 2-4). At week 16, when all animals showed a generalized thymic lymphoma, organ weights, lymphoid cells in thymus and spleen, relative T-cell numbers in thymus, spleen, and lymph nodes were significantIy elevated, whereas relative B-cell numbers were re- duced in spleen and lymph nodes (Figs. 1-6). One mouse of week 13 showed a thy- mic lymphoma, which preceded other lymphomas at this time; thereby the spleen was not infiltrated. The organ weight and the lymphoid cell numbers of the thymus were elevated, but splenic weight and lymphoid cell numbers did not indicate a lymphoma infiltration of this organ. However, immunocytologic shifts represented by a relative T-cell increase and a B-cell decrease - as for all Nbu mice of the 13th week - foreshadowed an impending splenic involvement. In this case, a low relative T-cell number of thymus was striking. Another Nbu mouse of week 13 showed al- ready grossly a splenic involvement by lymphoma. This finding was confirmed by organ weight and lymphoid cell number. Immunocytologically, a B-cell decrease and null-cell increase was remarkable. The T-ceil increase was thus comparable with other Nbu mice of week 13. An earlier splenic involvement of one mouse at week 11 was accompanied by an extreme B-cell reduction in spleen. In contrast to those mice in whom the spleen was involved by lymphoma, the null-cell prolifer- ation was more pronounced than the T-cell proliferation. Whereas the relative T- cell values of control mice, with regard to the organ distribution, showed thymus,

T-and B-Cell Determination in Various Lymphoid Tissues of Mice 157

100 % SPLEEN

5O

weeks

Fig. 3. Percental distribution of theta- and immonoglobulin-positive lymphoid cells in spleen. T cells: controls (o), Nbu mice (e); B cells: controls (~3), Nbu mice (m)

lymph node, and spleen in order of frequency in Nbu mice of week 16, T-cell values of about 80% were found in all three organs. At the same time, B cells in spleen and lymph node were reduced below 10%. During Nbu feeding and the transient thymic atrophy relative T-cell values did not exceed 45% in all investigated lym- phoid organs. Hence, the relative T-cell number of spleen was leading.

Discussion

A high lymphoma incidence was observed in young adult male C57BL mice fed the leukemogen N-nitrosobutylurea. The latency period amounted to 11 weeks. All lymphomas showed thymus involvement. Histologically, a poorly differentiated lymphoma of lymphoblastic cell type was seen. These data are consistent with the findings of others (Hiai et al. 1973). No lymphoma was observed in control mice during the period of investigation. Immunocytologic results in control mice com- monly corresponded well to data already described earlier. Only for T cells in the thymus are higher values reported by the majority of the authors for apparently methodological reasons (Lamelin et al. 1972; Rabellino et al. 1971; Raft 1969; Takahashi et al. 1971). Immunocytologic investigations in tumor-bearing mice re- vealed the T-cell origin of the induced thymic lymphomas. At week 16, when the lymphoma showed systemic organ involvement, the extraordinary rise of lymphoid cells in thymus above 220 x 106 and in spleen approaching 350 x 106 lymphoid cells

158 M. Kraus and G. R. F. Krueger

100' % LYMPH NODE

5O

weeks

I, B I'I 13 16

Fig. 4. Percental distribution of theta- and immunoglobulin-positive lymphoid cells in lymph node. T cells: controls (o), Nbu mice (o); B cells: controls (n), Nbu mice (I)

was accompanied by a similar T-cell increase in other organs. Only to an insignif- icant extent was the augmentation of lymphoid cells in spleen caused by a null-cell increase. Absolute B-cell values had even been decreased. Regarding relative lym- phoid cell numbers, the T-cell origin was indicated by a significant T-cell rise in thy- mus, spleen, and lymph nodes. Relative T-cell numbers above 80% in all investi- gated lymphoid tissues led to a suppression of relative B-cell numbers in spleen and lymph nodes. A significant null-cell proliferation at this time was not observed (Figs. 2-4). Considering these results, this Nbu-induced lymphoma can be classi- fied as a T-cell lymphoma ofthymic type with subsequent generalization. Two Nbu mice developed early thymic lymphomas with extensive spread to other organs as compared to other Nbu mice at the same age. In both cases, spleen has been in- volved by lymphoma growth. Within the spleen of one mouse of week 11 compris- ing 495 x 106 lymphoid cells, the null cells exceeded the T-cell fraction, whereas B- cell numbers were reduced. For another mouse with splenic lymphoma involve- ment a relative high null-cell value compared with Nbu mice of week 16 was deter- mined at week 13, although T cells were more frequent than null cells. An earlier lymphoma growth and generalization favored a higher null-cell value in the spleen as compared to T-cell numbers. It seems possible, therefore, that an acceleration of lymphoma development impaired the differentiation of T cells as indicated by the number of theta-antigen-carrying cells. It was absent from these null cells or at least reduced to an amount indetectable by the applied method. Conversely, the

T- and B-Cell Determination in Various Lymphoid Tissues of Mice 159

10 6 S

250

200,

150.

50

I

weeks 2 /,, 8 l i 13 16

Fig. 5. Lymphoid and theta-positive cell numbers in thymus. T cell: controls (o), Nbu mice (e); lym- phoid cells: controls (D), Nbu mice (w)

dignity of a thymic type lymphoma can be judged by the presence or absence of theta-andgen. A higher differentiation of the lymphoma cells suggests in this case not only a less reduced function, but also a slower lymphoma development and slower spread.

The usual lymphomas of the thymic origin after Nbu induction show a rela- tively high degree of differentiation as judged from their theta-antigen on the cell membrane. Their cells resembleimmunocytologicallyperipheral thymocytes or ma- ture thymic lymphocytes (Aoki et al. 1969). Other authors have found reduced cytotoxic anti-theta susceptibility on Nbu-induced lymphoma cells before manifes- tation of leukemia. However, in the early latency period no decrease of theta-posi- tive cells was observed (Hiai et al. 1973)i This difference to our data may be caused by the less sensitive immunofluorescence method as compared to cytotoxicity test- ing. The reduction of theta-antigen on a single lymphoma ceil implies a delayed dif- ferentiation of thymocytes either from stem cells or to mature thymus tympho- cytes. The changed H 2 antigen representation (Birch et al. 1975) would favor the former possibility, although a change in H 2 antigens on the surface of tumor cells may be caused by mutation as well. The loss of theta-antigen on cells of accelerated Nbu lymphoma development, however, argues for a differentiation state of lym- phoma cells between stem cell and thymocyte.

160 M. Kraus and G. R. F. Krueger

300"

250

50"

10 6 SPLEEN

I weeks 2 L 8 l i 1) 16

Fig. 6. Number of theta- and immunoglobulin-positive lymphoid cells in spleen. T cells: controls (o), Nbu mice (o); B cells: controls (rn), Nbu mice (n)

Between weeks 2 and 8 a thymus atrophy took place during Nbu administra- tion. Total thymus cells and absolute thymic T cells were reduced. As can be seen from relative T-cell distribution in thymus, most of the remaining cells did not ex- press the theta-antigen on the cell surface (Fig. 2). There was no indication of in- creased B-cell numbers. The effect of Nbu during the latent period can be explained by an inhibition of the differentiation from stem cells to thymocytes and mature T-lymphocytes. In addition, a reduced inflow of stem cells into the thymic cortex must be considered. This may be suggested by a reduced cellularity of the bone marrow described by other authors the latent period of a Nbu-induced T-cell lym- phoma and the reduction of absolute thymus cells (Fig. 1) (Hiai et al. 1973)�9

After the termination of Nbu administration a progressive T-cell increase indi- cated lymphoma development in thymus. There are two possibilities for a relative and absolute T-cell rise accompanying lymphoma development in thymus (Figs. 2, 5). T-cell lymphoma evolves either from a theta-positive cell that remained in the thymus during atrophy or from a theta-negative precursor comparable to some extent to normal T-cell development. The latter idea is supported by findings of atypical nests in the bone marrow earlier than in the thymus (Hiai et al. 1973). Besides, spontaneous and Nbu-induced thymic lymphomas of A K R mice irradi-

T- and B-Cell Determination in Various Lymphoid Tissues of Mice 161

ated lethally were derived from injected bone marrow cells and not from thymus cells applied at the same time (Shisa et al. 1977). Furthermore, the incidence ofmu- rine thymic lymphoma induced by a single injection of DMBA at birth is aug- mented by bone marrow cells additionally injected during the latency period (Ball 1968). Provided that the theta-positive tumor cell develops from a theta-negative precursor cell, it can be concluded that during lymphoma development a certain differentiation of tumor cells with regard to the expression of theta-antigen does occur. Furthermore, the relation of theta-positive/theta-negative cells increases during tumor development. Normal T-cell differentiation in thymus obviously is prevented by Nbu feeding. Therefore, maturation and proliferation are inhibited. Hence, there results a reduction of absolute T-cell numbers in thymus and a de- crease of the relation T cells/null cells. With lymphoma development following Nbu feeding T cell numbers increase absolutely and in relation to null cells. That indicates a "functional" lymphoma development along the pathways of normal T- cell differentiation. The question remains unanswered, at what time a breakdown of normal development within the T-cell system occurs, either caused by a prospec- tive impulse of growth which is suppressed as long as Nbu was fed or by a failure of growth control in a kind of an impaired feed-back mechanism, both induced by the long-lasting inhibition of T-cell differentiation and proliferation.

A similar growth behavior in a sense that the target cell of lymphoproliferative diseases is not identical with the proliferating tumor cell can be observed in mul- tiple myeloma of man. There exists a complete differentiation along the pathways of B-cell development to plasma cells with secretion of paraproteins, while a non- secreting B-lymphocyte is supposedly the target cell (Seligmann 1979).

More recent investigations of T-cell subpopulations (Batchelor 1979; Plata et al. 1979; Small 1979) provide information on the relationship of T-cell suppressor activity and differentiation within the T-cell system. Accordingly, less differenti- ated T cells exhibit tumor enhancement caused by an increased suppressor activity, while mature T cells possess rather tumor-inhibitory activity. The former is func- tionally dominating over the latter (Plata et al. 1979; Small 1979). Such an in- creased suppressor activity could be assumed for the observed T-cell lymphomas during early lymphoma development between weeks 8 and 11. A lower degree of differentiation is suggested by the distribution of theta-positive cells in thymus be- fore week 11 as described above, the presence of the enzyme "terminal deoxynu- cleotidyl transferase (TdT)" in N-nitrosomethylurea-induced T-cell lymphomas (Dexter et al. 1974; Saffhill and Chandhurt 1976), and the change of H 2 antigens on Nbu-induced lymphoma cells (Birch et al. 1975). An increased appearance of T suppressor cells can account for a lost growth control with T-cell proliferation. Therefore, more detailed studies using Ly-antigens to detect T-cell subpopulations are necessary during the latent period of lymphoma development.

A profound disorder of the T-cell system preceding lymphoma development was demonstrated by reduced values of theta-positive cells in thymus and lymph nodes which may explain the disturbed T-cell function. This could be associated with a reduced differentiation of cytotoxic T cells and/or an at least transitory in- crease in T suppressor cells inhibiting cytotoxic (killer) T cells (Meruelo and Mc Devitt 1978; Plata et al. 1979; Schirrmacher and Shantz 1979; Small 1979). This mechanism supports the thesis of an immunofunctional lymphoma development.

162 M. Kraus and G. R. F. Krueger

Krueger et al. succeeded in inducing malignant lymphomas by continuous admin- istration of non-carcinogenic antigens and synchronous immunosuppression (Krueger 1971, 1972; Krfiger et al. 1973; Zoltowska 1975). Thereby, theta-positive lymphoblastic lymphomas of thymic type were observed. In these animals T-cell response was completely abolished (Krueger 1971, 1972; Krueger et al. 1973). It was argued that there existed a stimulus of proliferation caused by the application of antigen and a concurrent inhibition of differentiation within lymphatic tissue re- leased by an immunosuppressive drug. Furthermore, a neutralization of antigen was lacking because of an insufficient immune response that supported continuous proliferation and led to malignant lymphoma growth (Krueger 1971).

Preliminary studies with Nbu fed in a non-leukemic dose to older mice showed similar changes concerning thymic weight, cell number, and T-cell distribution during Nbu administration. However, changes were not as prominent as described in the present paper and returned to control values after Nbu-feeding had been ter- minated. Selective immunofunctional investigations revealed a reversible sup- pression of cellular and humoral immunity during Nbu administration (Kraus 1980). In the present study T-cell development in the thymus was disturbed, T-cell numbers in the spleen were reduced, and alternating variations in number of un- differentiated and more differentiated cells of thymic origin accompanied by nor- mal B-cell values occurred. A normal peripheral T-cell population was not main- tained numerically.

Although a T-cell lymphoma developed after Nbu-feeding, also B-cell values were affected during latency period. The decrease of lymphoid cells in spleen dur- ing Nbu administration was mainly evoked by a reduction of B cells (Fig. 6). Thus, a suppression of humoral immunity could be expected as well. As mentioned by other authors there is a marked decrease in cellularity of the bone marrow during the latency period of Nbu-induced thymic lymphomas (Hiai et al. 1973). This could add to an immunologic defect. The lack of stem cells of bone marrow origin cer- tainly could influence the defect of the T-cell system, but besides that a differenti- ation block in thymus must also have taken place.

Taken together, early immunocytologic changes during the latency period of lymphoma development indicate a general immunosuppression before the onset of lymphoma development. Other authors have already described an irreversible in- jury of immune function beginning at the start of Nbu administration (Yokoro et al. 1973). The crucial influence of immunosuppression on chemically induced lym- phoma growth was also demonstrated by the fact that a single non-leukemogenic dose of Nbu causes malignant lymphomas when combined with an immunosup- pressive agent (Imamura 1973).

Involvement of oncogenic viruses in Nbu-induced lymphoma development, e.g., by an activation of an endogeneous viral genome, has been discussed (Ima- mura 1973). A virus similar to Gross virus was considered as the agent. A compa- rable viral involvement could not be established for the Nbu induction of malig- nant lymphomas in rats. These are known to have a lower natural viral contami- nation than mice (Imamura 1973). Other studies indicate that infectivity of murine leukemia virus is not necessarily correlated with the induction ofleukemias by Nbu or DMBA (Kawamura 1976; Odaka 1975). In mice with a low rate of spontaneous leukemias including C57BL which have been characterized as G antigen negative

T- and B-Cell Determination in Various Lymphoid Tissues of Mice 163

(Kaplan 1967; Stockert et al. 1971), genetic material of murine leukemia virus has been found (Aaronson et al. 1971; Rowe et al. 1972). However, C57BL mice strongly resistant to the infection passage A virus normally possess a G antibody titer which suggests a sufficient immunity against natural G virus infection. This could be abolished by the immunosuppressive effect of Nbu. An activation of en- dogeneous viral material may possibly follow. For example, AKR mice with a high spontaneous leukemia rate do not exhibit G antibodies (Aoki et al. 1968) and thus may exhibit a defective natural immunity against the respective virus.

An isolated immunosuppressive effect appears not sufficient for lymphoma in- duction. A stimulus of proliferation probably acts upon the lymphatic tissue in ad- dition, which again taken alone does not cause malignant lymphoma (Krfiger 1971). The impulse of proliferation in chemical leukemogenesis could be mediated by non-leukemogenic environmental antigens or activated endogeneous viruses which are normally controlled by the immune system and activated by im- munosuppression. The leukemogen itself, respectively a metabolic agent, possibly exhibits antigenic stimulation establishing proliferation.

After Nbu administration, a T-cell lymphoma developed with a high incidence. T-cell proliferation started in the thymus at week 11 and was followed by the T-cell increase in spleen and lymph nodes. Thus, from immunologic data there is no doubt that the onset of T-cell spread originated from the thymus. A prominent T- cell rise in lymph nodes occurred when lymph nodes were generally involved in lymphoma growth at week 16.

Though virus-induced thymic lymphomas often exhibit T-cell properties, null- cell lymphomas have also been observed (Krueger, 1977; Mertens and Krueger 1976; Shevach et al. 1972). Compared with a Moloney virus-induced thymic lym- phoma which has been investigated immunocytologically (Mertens and Krueger 1976), differences were obvious. Thus, a null-cell proliferation characterized the generalization of thymic lymphomas in spleen and lymph nodes. Theta-positive and immunoglobulin-positive cells disappeared progressivly. A less differentiated tumor cell probably of stem cell origin accounted for the malignant proliferation and differed from the theta-positive Nbu lymphoma cell. It was interesting to note that the early Nbu-induced lymphoma appeared less well differentiated also with regard to theta antigens, and null-cell proliferation was prominent in one case. The increasing theta positivity of tumor cells during advanced lymphoma growth sup- ported this observation and additionally favored the thesis of immunofunctional lymphomagenesis. Radiation-induced thymic lymphomas, such as Nbu lym- phomas, were found to be theta-positive (Haran-Ghera et al. 1975). However, in contrast to Nbu-induced thymic lymphomas H 2 representation was increased (Birch et al. 1975; Haran-Ghera et al. 1975; Meruelo and Mc Devitt 1978). Theta positivity and strong H 2 representation allow to compare these cells with mature thymus lymphocytes (Aoki et al. 1968) and point toward a higher degree of differ- entiation of cells in radiation induced leukemia than in Nbu-induced T-cell lym- phomas. In essence, thymic lymphomas induced by different agents may roughly be classified according to their state of differentiation. Of the mentioned thymic lymphomas, the radiation-induced lymphoma represented the highest and the Mo- loney virus-induced lymphoma the lowest state of differentiation. An intermediate position is occupied by Nbu-induced T-lymphoma cell. However, considering the

164 M. Kraus and G. R.F. Krueger

d y s f u n c t i o n a l t h e o r y o f l y m p h o m a d e v e l o p m e n t this c o i n c i d e n c e m a y p r o b a b l y

v a r y a c c o r d i n g t o h o s t c o n d i t i o n s , such as age a n d t h y m i c ac t iv i ty a t the t i m e o f

l y m p h o m a induc t i on .

Acknowledgement. We wish to thank Dr. H. Shisa, Saitama Cancer Center, Japan, for his helpful suggestions during the course of the experiment. Moreover, we are indebted to Ms. B. Koch and Mr. B. Raftery for technical help.

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Received December 8, 1980/Accepted March 11, 1981