Immunologic unresponsiveness induced by cytosine arabinoside in adult mice

JOURNAL

OF SURGICAL

RESEARCH

Immunologic

16, 449-456 (1974)

Unresponsiveness Arabinoside

ALLYN

G. MAY,

M.D.,

BERNARD

in Adult

PANNER,

M.D.,

CYTOSINE ARABINOSIDE (CA) has been observed to promote immunologic unresponsiveness in adult mice when used in conjunction with the administration of cellular antigen [ 11. For the C3H/CBA strain combination the most effective protocol was found to be: Adult

Mice AND

EK daay

by Cytosine

MARK

W.

EASTLAND,

M.D.

greater than 38 days or in a larger fraction of mice showing prolonged unresponsiveness. The absolute requisites for expression of this effect were found to be: (1) administration of the cellular antigen prior to administration of CA, and (2) treatment of the mice with both antigen and CA. In an

9 CBA mice ~F Hybrid 1y;phocytes onday -10

Induced

T C3H skin graft day zero

dn -;, ,

MST 22-38 days. 21-27s of grafts not rejected

Protocol

The median survival time (MST) of these grafts was prolonged to 36-38 days, whereas untreated controls rejected grafts with an MST of 13-14 days (Table 1). Twenty-seven percent of grafts enjoyed markedly prolonged survival showing no rejection as long as observed for up to 125 days (Figure 1). Second C3H skin allografts were readily accepted by mice carrying long-standing primary C3H grafts. The CA, as used in this Protocol, caused no untoward effects in the mice. Continued administration of the drug for longer than three days gained no additional prolongation of graft survival. Prior splenectomy did not prevent prolongation of graft survival. Many variations of this treatment schedule were studied. None resulted in an MST

effort to define additional characteristics of this phenomenon further experiments were performed: 1. To determine the specificity of the phenomenon and the influence of histocompatibility differences. 2. To attempt prolongation of graft survival passively by means of plasma from Protocol mice. 3. To determine the effect of the adoptive transfer of immunity on enduring allografts. 4. To determine the influence of a prior state of immunity on the phenomenon. 5. And to discern any histologic changes in the lymphoid tissues in mice treated according to the protocol.

From the Departments of Surgery and Pathology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. Supported by a grant from the New York State Kidney Disease Institute, Albany, New York. Submitted for publication December 24, 1973.

Mice were obtained from the Jackson Laboratories or derived from Jackson Laboratory stock. All recipients were adult Q CBA (H-2k). Skin donors were o C3H (H-2”), o Ajax (H-2”), or o AKR (H-2k).

MATERIALS

449 Copyright ~11 rights

@ 1974 by Academic of reproduction in any

Press, 1~. form reserved.

AND

METHODS

450

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Table 1. Specijicity Group

No. mice

A B C 11 E F G II I

9 17 14 20 13 20 17 1Q 19

RESEARCH,

and Injluence

Type of lymphocyte used in protocol

Skin donor

None X C3H)F, X CSII)F1 None None 3 CBA None (CBA X Ajax)Fl (CBA X Ajax)Fl

C3H C3H AKR AKR 07 CBA 8’ CBA Ajax Ajax Ajax

(CBA (CBA

a-•C3H+TREATED CBA MST20DAYS CBA MST15 DAYS oC3H-WNTREATED

DAYSAFTERGRAFTING Fig. 1. The MST of grafts on mice treated with cytosine arabinoside and F, hybrid cells was extended to 20 days compared to 15 days for control mice. Moreover, 27% of mice of the cytosine group kept their grafts longer than 125 days.

In experiment II male C3H skin donors were also used. F, hybrids were male and female bred from CBA X C3H or CBA X Ajax matings in the University of Rochester Vivarium. Suspensions of F, hybrid lymphocytes were prepared from spleens and peripheral lymph nodes of equal numbers of male and female mice freshly sacrificed by cervical dislocation. The cell concentration was adjusted to 200 X 106/0.5 ml in Hanks’ medium. Suspensions of sensitized lymphocytes were obtained from spleens and lymph nodes of CBA mice which had rejected C3H skin grafts at least 1 week before. The cell concentration of the suspension of sensitized lymphocytes was adjusted to 250-300 X 10’/0.5 ml in Hanks’ medium. Administration of the lymphocyte suspensions was by means of tail vein injection after intraperitoneal injection of 50 IU of aqueous heparin. The cytosine arabinoside

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5,

Differences MST (days)

Schedule of CA treatment None -8, -8, None None -9, -8, None -9, -8, -10, -9, -9, -9,

MAY11974

-7 -7

-7 -7 -8

13-14 36-38 16-17 13-14 38% rejected by day 130 None rejected by day 130 9 9-10 Q-10

(CA)” was prepared in distilled water at a concentration of 1 mg/ml. The drug was administered intraperitoneally in doses of 40 mg/kg/mouse/day for 3 days. The grafting techniques utilized fullthickness skin of approximately 180 mm” fixed to the panniculus carnosus of the recipient by plaster casts. Casts were removed on day 7 and the grafts were observed daily, when possible, for rejection. For enduring grafts less frequent observations were made. The criteria for rejection included graft contraction, eschar formation, loss of graft hair, or dissolution of the graft. The end point of rejection occurred tissue was grossly when no donor discernible. Experiment I. Specificity of the Phenomenon and Influence of Histocompatibility Differences The specificity of the phenomenon was tested by means of two groups of CBA mice, using (C3H X CBA) F, cells in each, but grafting one with C3H skin (Group B) and the other with AKR skin (Group C). The degree of histocompatibility in these combinations is similar and differences in the MST of the respective grafts can be attributed to immunologic specificity. In order to determine the influence of differences in histocompatibility on the expression of this phenomenon, the weak, male-to-female, CBA combination (Groups * Cytarabine

(Upjohn).

MAY,

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AND

EASTLAND:

IMMUNOLOGIC

E and F) and the strong, H-2 differing combination, Ajax to CBA (Groups G, H, and I), were submitted to appropriate modifications of the protocol (Table 1). Experiment II. Plasma Transfer Experiments (Table 2) The plasma for transfer experiments was obtained at days 10, 15, and 21 by bleeding protocol mice from the retroorbital sinus. Plasma was also obtained from Protocol mice which were bearing healthy C3.H skin grafts at or beyond day 37. The plasma specimens were frozen and stored at -90°C until used. Skin grafts from normal C3H mice were incubated at 20°C for 15 min with the protocol plasma. After incubation the grafts were placed on normal CBA recipients, each receiving 0.15-0.20 ml of the remaining respective plasma by injection intraperitoneally (Groups J, K, L, and M) . Control CBA mice were treated similarly, receiving C3H skin grafts incubated in normal CBA plasma and receiving intraperitoneal injection of normal CBA plasma (Group N) . The grafts were observed from day 7 to completion of rejection. Experiment Immunity

III.

Adoptive

Transfer

of

Protocol mice which bore healthy skin grafts for 37 days or longer received 250-300 X 106 sensitized isogeneic lymphocytes via the tail vein. Control mice, also bearing healthy grafts, received F, hybrid cells in the same manner. The grafts of each group were observed for rejection from day 7. Table 3. InJluence

Group

No. mice

0 :

16 13 12

R

16

Previous

treatment

None C3H skin graft Protocol Protocol

UNRESPONSIVENESS

Table 2. Plasma Group

No. of mice

J K L M N

5 ; 6 12

Experiment (Table 3)

Transfer MST (days)

Plasma lo-Day Is-Day al-Day 37-Day Normal

451

Protocol Protocol Protocol Protocol CBA plasma

13 12-13 14-15 14 14

-

IV. The Influence of Immunity

Normal female CBA mice (Group P) which had rejected C3H skin in first-set fashion, were submitted to the Protocol and their Protocol grafts were observed until rejected. Other groups of CBA mice were (Group Q) subjected to the Protocol and, after their Protocol C3H grafts rejected, were simply grafted with C3H skin again; and (Group R) were submitted to the Protocol twice and, having rejected their first Protocol grafts, the second Protocol skin grafts were observed until rejected. The MST’s of these groups were compared to that of Group 0, a standard Protocol group. Experiment V. Histologic phoid Tissues

Changes in Lym-

Lymphoid tissues, with the exception of bone marrow, were obtained for gross and histologic study by sacrificing groups of mice at intervals during the Protocol treatment and up to 30 days after grafting. Sections were evaluated by light microscopy by B.P. who broke the key after completion of his examination. Control groups, which received only F, hybrid cells or CA, were also studied. of Immunity Last treatment

MST (days)

Protocol Protocol C3H skin graft Protocol

22 9 9-10 10-11

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RESEARCH,

[CBAxC3H)F, CELLSINJECTED

---\

-i

5t~51TiZp CBA CELLSINJECTED.

@oiTrn,-----J 0

5

IO 15 20 25 30 35 40 45 50 55 t0

I

65

70 75

DAYS AFTERCELL INJECTION

Fig. 2. Long-enduring grafts on cytosine-treated mice were rejected after the adoptive transfer of immunity by intravenous injection of sensitized isogeneic lymphocytes. The grafts of animals receiving indifferent F1 hybrid lymphocytes were not rejected.

Statistical analysis of the skin graft survival data was carried out by means of the Kruskal-Wallis rank test and Fisher’s exact test (See Appendix) [2, 31. RESULTS 1. Specificity of the Phenomenon and InfluDifferences ence of Histocompatibility (Table 1) In the AKR-to-CBA combination, involving a weak histocompatibility difference similar to that of the C3H-towith combination, treatment CBA (CBA X C3H) F, hybrid cells and CA did not result in significantly prolonged survival of the AKR skin grafts (Group C) compared to C3H grafts (Group B). Thus, the unresponsiveness induced by the Protocol is specific and dependent on the alloantigens represented on the F, cells. It proved impossible to extend the MST of the H-2 incompatible strain combination, Ajax to CBA (Group G) , by means of two modifications of the Protocol each of which used (CBA X Ajax)F, hybrid cells (Groups H and I). However, survival of all the male skin grafts on the Protocol female CBA mice (Group F) was prolonged remarkably beyond the gra.fts of control Group E.

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2. Plasma Transfer Experiments (Table 2) None of the mouse groups receiving skin grafts pretreated with Protocol plasma and intraperitoneal Protocol plasma showed any evidence of prolonged graft survival. All grafts were rejectable by day 18 and the MST of each group (J, K, L, and M) was not significantly different from that of the control Group N. 3. Adoptive

Transfer

of Immunity

The six Protocol mice, carrying healthy C3H skin grafts and injected intravenously with sensitized isogeneic lymphocytes, rejected their grafts on day 9-76 after cell injection (Fig. 2). Six control mice, carrying healthy grafts, received intravenous F, hybrid lymphocyte injections. Their grafts showed no evidence of rejection for as long as observed (71-126 days). 4. Influence (Table 3)

of

Pre-existing

Immunity

In mice (Group P) which had previously rejected C3H skin grafts, the Protocol faiIed to prevent a second-set response Two groups, in which mice had been subjected to the Protocol and subsequently rejected their Protocol grafts, were regrafted (Group Q) with C3H skin or submitted a second time to the Protocol (Group R) . In each group the MST was shortened from that expected for the C3H-to-CBA combination (MST = 13-14 days), although a few grafts survived beyond 34 and 56 days (Fig. 3). Application of the Kruskal-Wallis rank test indicates that the survival of grafts of Groups Q and R compared to those of Group P was significantly different. 5. Lymphoid

Histology

Figure 4 shows the peripheral leukocyte response in three groups of adult CBA mice which received F, cells alone, CA alone, and the combination of F, cells followed by CA as prescribed by the Protocol. Injection

MAY,

PANNER

AND

EASTLAND

: IMMUNOLOGIC

of F, hybrid cells was followed within 24 hr by peripheral leukopenia and lymphocytopenia. With the administration of CA, the peripheral counts rose but not to pretreatment levels. Spleen and peripheral lymph node weight indices (i.e., weight of organ/total body weight) of groups of mice, sacrificed at intervals during treatment with F, cells and CA, showed no significant change. Nor did microscopic examination of sections of these tissues show any morphologic changes of significance. However, the thymus showed consistent changes characterized by a 26% increase in thymus weight index after injection of the F, hybrid cells followed by a fall to 35% of its pretreatment weight index with administration of CA. The thymus weight index gradually returned to normal over the course of 30 days (Fig. 5). Microscopically the thymuses showed a temporary marked cellular depletion. DISCUSSION Cytosine arabinoside has antiviral, antitumor, and immunosuppressive properties. The latter quality is not marked in the murine species. However, its ability to extend the survival of murine allografts when used with injection of cellular antigen is significant and worthy of study. The phenomenon resembles tolerance. The unresponsiveness is specific for the strain of cell used in pretreatment. The efficacy of the Protocol in prolonging skin a!lograft survival is directly proportional to the degree of histocompatibility of the several strain combinations described. Rejection of long-surviving allografts can be brought about adoptively by transfer of presensitized isogeneic lymphocytes. No evidence for enhancement was detected in plasma transfer experiments. Finally, it was not possible to overcome an established immunity. However, some Protocol mice, which rejected their Protocol allografts, kept secondary allografts for unusually long periods of time (Groups Q and R).

453

UNRESPONSIVENESS

VJ s2loo 80

-. pR01lx01 c---d PROTOCOl+2nd CW SKIN P--* PROTOCOL+PROTOCOL -SENSITIZED C@+I+~OTocOL

z,,$I!?t,

DAYSAFTERGRAFTING

Fig. 3. In spite of having rejected a previous skin graft, protocol mice retreated with F1 hybrid lymphocytes, cytosine arabinoside, and a second graft did not invariably reject their second grafts in an accelerated fashion.

CELLS

CELLS

Fig. 4. The peripheral leukocyte response of mice treated with F, hybrid cells and cytosine arabinoside was not different from the response of controls receiving cells alone or cytosine arabinoside alone.

CELLSAND CYTOSINE 09 0'

0

1

F, HYBRIDCEllSt !NlECTEDI"

K

2

3

4

5

6

7

8

9 IO TIME DAYS tsKlN GRAFT

ARABINOSIDE INJECTEDIP

Fig. 6. The thymus weight index changed most markedly in mice treated with F, hybrid lymphocytes and cytosine arabinoside.

This suggests that the activation of spite of the history The expression

the treatment prevented immunologic memory in of graft rejection. of unresponsiveness only

454

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in a fraction of the CSH/CBA Protocol mice seems to be related to the histocompatibility difference involved inasmuch as it is completely expressed in the weak, sexdifferent combination, male CBA to female CBA, and not expressed at all in the H-2 different combination, Ajax to CBA. In view of the pooling of equal numbers of male and female F, hybrid cells as the source of cellular antigen, each female CBA recipient was pretreated with cells of the same sex distribution. Differences in quantity of sex-linked histocompatibility antigen would not seem to be a possible cause for the fractional expression of unresponsiveness which was observed. Previous experiments in rats have shown thymic atrophy in response to treatment with cytosine arabinoside [4]. The present experiment demonstrates this in mice and also shows that intravenous injection of F, hybrid cells causes transient thymic enlargement. It is interesting to speculate whether these thymic changes may be manifestations of the mechanism of selective prolongation of graft survival. Perhaps the clone of thymic cells, which is necessary for rejection of the graft, is stimulated by the introduction of F, hybrid cells, and, in this stimulated state, is selectively susceptible to killing or to modification by the subse-

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quent administration of cytosine arabinoside. This theory could be tested by determining whether transplantation of thymic tissue from such selectively unresponsive mice to neonatally thymectomized isogeneic recipients would result in the transfer of specific unresponsiveness. SUMMARY Cytosine arabinoside when used in conjunction with F, hybrid lymphocyte injection extends significantly the median survival time of C3H skin grafts on CBA recipients. Some grafts (27%) show little evidence for rejection over long periods of time. This treatment could not overcome a preexisting specific immunity. However, some of the treated mice, having rejected Protocol grafts, failed to evelop specific immunity to secondary grafts. The thymus was observed to respond to the F, hybrid cell injection and cytosine arabinoside by enlargement followed by atrophy and cellular depletion. The phenomenon is speculated to be a state of drug-induced tolerance. APPENDIX Analysis

of Skin Graft Data

(1) Kruskal-Wallis rank test for difference between group means of B, A, C, and D, H = 20.931 (significant at 5% level). P 2 0.001 Group B A C D

Size 17 9 14 20 x&.95)

Groups to be compared *B - D *B - A B-C C-D C-A A-D

[Bi -

Ttjj

24.772 21.48 11.433 13.339 10.047 3.292

Mean rank 44.647 23.167 33.214 19.875 = 7.81

Xs2(.95) . “(“1:

‘)

16.0987 20.1195 17.614 17.007 20.852 19.59

MAY,

PANNER

Kruskal-Wallis

AND

EASTLAND

test for B and C alone H = 8.922 Group B c

(2) Comparison

Size 17 14

455

UNRESPOSSIVENESS

P = 0.001 Mean rank 20.412 10.643

of F and E No. mice rejecting the graft

No. mice which did not reject

0 5 5

20 8 28

F E

Probability

: IMMUNOLOGIC

20 13 33

00 __

that no graft is rejected with F treatment

200

=

13 5

5!8! 13!

=33! 0 335

= .0056

5!8!

We reject the hypothesis that the two treatments F and E do not differ at 5% level. (3) Kruskal-Wallis rank test for difference between group means of H, G, and I is H = 2.430; and xz2(.95) = 5.99. P = 0.303. Hence we conclude that the differences in groups means are not significant. [Here Prob (H > 2.430) = .3]. (4) Comparison of group means for J, K, L, M, N (different serums). Kruskal-Wallis rank test, H = 11.736; xJ2(.99) = 13.3 and xa2(.95) = 0.49. There is no significant difference at 1% level. P = 0.011. [P(H 2 11.736) = .02] Group J L N M K

Size 5 9 12 6 5 37

Mean rank 29.1 23.222 18.708 12.167 10.2

Groups to be compared J-K J-M J-N J-L L-K L-M L-N N-K N-M M-K

18.9 16.94 10.40 5.88 13.02 11.055 4.514 8.508 6.541 1.967

21.09 20.19 17.75 18.60 18.60 17.57 14.70 17.75 16.67 20.91

Hence we conclude that all pairwise differences are insignificant.

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(5) Influence of immunity: (comparison of 0, I’, Q, R) Kruskal-Wallis rank test, H = 26.618. The groups are significantly different since xs2(.95) = 7.81. P z 0.001 Mean rank

Group 0

Size 16

42.62.5

Q

12

29.958

IL 1’

16 -13 57

29.156 11.154

Critical const’ant Group to be compared

IR - Rji

0-Q

12.667

O-R 0 Q*Q “R -

1’ R P P

13.469 31.471 so2 18. so4

18.002

ACKNOWLEDGMENT Miss Mary Ann Wanes is to be congratulated for her successful construction of the manuscript.

REFERENCES 1. Amaku, E. O., May, A. G., Hall, T. C., and Linke, C. A. Prolongation of mouse skin allograft survival by treatment with cytosine arabinoside in combination with F-l hybrid

-(;

+ ;)]l”

17.71 16.40 17.32 17.71 18.569 17.32 lymphocytes. Transplantation 12:249-252, 1971. theory and meth2. Brownlee, X. A. Slatistical odology in science and engineering. Wiley, New York, 1965. 3. MiIler, R. J. Simultaneous statistical inference. McGraw-Hill, New York, 1966, 4. Gray, G. D., and Mickelson, M. M. The immunosuppressive activity of adamantoyl cytarabine III. Immunosuppressive specificity in rats. Immunology 19:417-428, 1970.