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The mechanism of immunological tolerance in mice treated with cyclophosphamide. The study found that specific tolerance lasted for up to nine months in some animals and required repeated administration of sheep red blood cells (SRBC) for maintenance. The smallest number of SRBC required for tolerance induction was 107. The document also discusses the role of antigen in inducing and maintaining tolerance in drug-treated animals. The study concludes that drug-induced immunological tolerance differs significantly from both pneumococcal polysaccharide paralysis and classical, acquired tolerance.
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ON THE MECHANISM OF IMMUNOLOGICAL
TOLERANCE IN CYCLOPHOSPHAMIDE-TREATED MICE
Boston, Massachusetts
(Received 28 July 1969)
SUMMARY
could elicit (^) antibody synthesis within (^5) days in normal (^) mice. Since the effects of
that the mechanism of tolerance induction involved destruction of antigen- stimulated cells. In support of this is the finding that mice treated with small doses of cyclophosphamide were rendered tolerant only when SRBC were given before the drug. Drug-induced immunological tolerance thus appears to differ significantly from both^ pneumococcal polysaccharide paralysis and classical, acquired tolerance. A central loss of (^) immunocompetence does not occur in the former, while the latter requires for its induction (^) the administration of antigen in a dose or form that does not stimulate antibody synthesis.
Drug induced^ immunological tolerance is an antigen-specific deletion of immunological responsiveness that results from treatment with (^) cytotoxic agents. Many examples of this
obscure. The role of the chemotherapeutic agent in bringing about tolerance has been emphasized frequently, but the (^) equally important function of antigen in inducing and
Correspondence: Dr^ Robert S. Schwartz, Clinical Immunology Service, New England Medical Center Hospitals, 171 Harrison Avenue, Boston, Massachusetts 021 11, U.S.A.
87
88 Amira Many and R. S. Schwartz
maintaining tolerance in drug-treated animals is not well^ appreciated.^ In^ this paper^ we will present evidence that in drug-induced immunological tolerance there^ is, as in^ acquired immunologic tolerance, a central loss of^ specific^ immunological^ reactivity. However,^ the induction of tolerance in^ cyclophosphamide-treated^ mice^ requires^ an^ amount^ of^ antigen that can stimulate antibody synthesis in normal^ animals.
Animals
C57BL/6J mice, aged 8-10 weeks, were housed in plastic cages and had free access to water and a standard mouse diet (Purina).
Antigens
Sheep red blood cells (SRBC) (Colorado^ Serum^ Co.)^ and^ horse^ red^ blood cells^ (HRBC) (a gift of the Massachusetts Public^ Health Biological^ Laboratories)^ were^ washed three times in 0 15 M^ saline and^ resuspended^ to^ the^ desired^ concentration^ in 0^ 15 M^ saline.^ Unless stated
of 20 volumes^ °/%) injected intraperitoneally.
The drug was dissolved in 0-08 M saline and administered intraperitoneally in^ concentra- tions of from 1-5 to 3 0 mg/ml, depending upon the experimental design.
Spleen cell suspension
was used throughout.
X-Irradiation
within several minutes, they received 5 x^108 SRBC.^ Every 5 days thereafter^ they were^ given
Amira Many and R. S. Schwartz
5 x 108 SRBC. Another group of twenty mice^ was treated^ in the same way^ except^ for the injection of cyclophosphamide. Mice of both^ groups were bled^ every^5 days, just^ before injection of the SRBC.^ The^ experiment^ was^ continued^ for^ about 9^ months.^ The^ agglutinin titres found in^ the^ drug-treated^ animals^ were^ always^ less^ than^ those^ of^ the^ control^ mice (Fig. 1). During the first 4 months of the experiment, responses to SRBC by the cyclo-
the mice in the control group responded to the antigen; the fraction of cyclophosphamide- treated mice forming antibodies to the SRBC rose gradually (Fig. 2), but never reached
serum) was linear during the first 5 months; thereafter, a^ small, constant^ fraction^ of the
14-
12
10 X
@ 8 _ c, 0
o 0
a-A - 0 0 20 4 60 8 10
FIG. 3. Specificity of drug-induced immunological tolerance. The experiment was carried out as in Fig. 1; beginning on day 5 control and experimental animals were given HRB3C every 5 days. (^) 0r, Control; (^) *, experimental; *, response to sheep red blood cells.
Tolerance specificity was determined by challenging the cyclophosphamide-treated and control mice with a second antigen, HRBC. Five days after the first injection of SRBC, the mice were challenged with 5 x 108 HRBC. This was repeated every 5 days. Fig. 3 shows that the cyclophosphamide-treated mice did not differ from those of the control group with respect to their responsiveness to HRBC.
The central loss of^ immunocompetence Tolerance was induced in mice by the method just described, and 20 days later, or 5^ days after the fourth injection of SRBC, the number of plaque forming cells^ (PFC)^ in^ their spleens was enumerated. Two^ groups^ of mice^ were^ studied: in one, the^ initial dose^ of SRBC
reduced numbers in the drug-treated animals (Fig. 4). The^ numbers^ of mice are small, but
than 5 x 108 SRBC, was more tolerogenic in cyclophosphamide-treated mice.
90
Immunological (^) tolerance in mice 91
suspensions of their spleen cells were injected into irradiated mice. The (^) recipients were (^) given
100,000 _ 5 x10SRBC (^5) x ISRBC Direct Indirect^ Direct Indirect plaques plaques^ plaques^ plaques
10,
a-
1,
FIG. 4. PFC in (^) normal and drug treated mice. All groups of (^) animals received injections of SRBC every (^5) days, and splenic PFC were estimated 5 days after (^) the fourth dose of SRBC. Open columns, controls; (^) hatched columns, drug treated mice. Eight animals (^) per group.
TABLE 1. Specific, adoptive tolerance of SRBC in (^) irradiated recipients of spleen cells from mice with (^) drug-induced tolerance of SRBC
Repopulation by spleen cells (^) from: Days after irradiation (^) Normal mice Tolerant mice
Anti-SRBC (^) Anti-HRBC Anti-SRBC Anti-HRBC
(^10) 2-6±3-0 (^0) 0-3±0-8 1-0+2- (^15 5) 3±1-2 70+1-7 0-3+0-8 (^) 6-8±1- (^20) 11-0±3-0 11-0±3-0 0-8±1-5 10-8+1- 25 12-3+1-0 (^) 1-0±1-
tolerant or normal (^) mice. Five days later all receipients were (^) injected with a mixture of 5 x (^108) SRBC+5x 108 HRBC. These (^) antigens were given every 5 days (^) thereafter. The
Immunological tolerance in mice 93
Fig. 5 shows that the prolonged tolerance in cyclophosphamide-treated mice was^ not due to persistence of^ non-specific drug injury.^ Recovery from cyclophosphamide by^ 120 hr in the absence of additional antigen was demonstrated by studying serially the immune
10 to_
8;
0~~~~~~~~
6 I
Days before or otter cyclophospharnide FIG. 7. Sensitization or tolerance induction in mice given SRBC before or after cyclophos- phamide. Horizontal stippled bar shows the standard deviation of anti-SRBC titres of mice not given the drug. Eight to ten mice per group.
The differences in the number of splenic PFC formed by these two groups is significant at each point studied. This indicates that although cyclophosphamide is of undoubted im- portance in making possible the induction of immunological tolerance, antigen must be present if the drug is to exert an immunologically specific effect.
Requirement for an immunogenic stimulus The smallest number of SRBC needed for the induction of immunological tolerance in
107 or 108 SRBC, together with 150 mg/kg cyclophosphamide. Control animals received no drug. Every 5 days thereafter all mice received 5 x 108 SRBC. Tolerance developed only in those groups of drug-treated mice that received an initial dose of antigen capable of provoking an immune response within five days in normal mice (Fig. 6). The smallest tolero- genic dose of antigen was 107 SRBC. A 'low zone' of tolerance induction was not detected.
Inhibition of antigen-selected cells SRBC were injected at different times before or after a single dose (150 mg/kg) of cyclo- phosphamide. Every 5 days after the first dose of antigen^ the various groups of mice received 5 x 108 SRBC. Haemagglutinin titres were measured on day 20. Tolerance was induced when antigen was given any time from -72 to + 24 hr before or after the drug (Fig. 7), but it was not possible to induce tolerance when antigen was given 48 hr or more after, or 96 hr or more before cyclophosphamide.
Amira Many and R. S. Schwartz
06
0 4
2 0
0~~~~~~~
0 25 50 75 100 125 150 Dose of cyclophosphamide (mg/kg) FIG. 8. Sensitization or tolerance induction in mice given different doses of cyclophosphamide before or after 5 x 108 SRBC. Horizontal stippled bar shows the standard deviation of anti- SRBC titres of mice not given the drug. Six to eight mice per group. *, Cyclophosphamide on day 0; o, cyclophosphamide on day + 1.
2 5 x 10' SRBC
10
8
4
2
0
0 10 20 30 40 50 Days FIG. 9. Tolerance or sensitization in mice given cyclophosphamide, 75 mg/kg, either^ simultan- eously with (@) or 1 day after (A) 5 x^108 SRBC.^ o, No^ cyclophosphamide.
94
96 Amira Many^ and R. S.^ Schwartz
Specificity of the maintenance^ antigen^ was determined^ in^ a group^ of mice that was^ given HRBC instead^ of SRBC^ every^5 days after^ an inducing^ injection of SRBC. The^ results (Fig. 12) show that only mice maintained with SRBC were persistently tolerant^ of^ that antigen.
18 1
15
12-
6-
5 x 106 SRBC I l 0 20 40 60 80 Days FIG. 1 1. All mice were given 5 x^108 SRBC on day 0. The^ 'maintenance'^ group (A)^ received SRBC every 5 days thereafter during the entire^ experiment.^ Antigen^ was withheld^ from the other two groups from day 0 to day 30; it was then given every^5 days.^ Eight^ to ten^ mice per group. a, No maintenance; *,^ no^ cyclophosphamide.
DISCUSSION
tolerance. The first is that in cyclophosphamide-treated mice there^ is^ a^ central loss of
Immunological tolerance in (^) mice
ance to heavily irradiated mice. The tolerance in these mice thus differs (^) significantly (^) from the paralysis induced by pneumococcal (^) polysaccharide, in which (^) antibody-forming cells are present but their product-antibody-is apparently absorbed (^) by persisting antigen (Howard et (^) al., 1969).
8 f
7-
6 -
(^2) --1 T
004
10 20 30 40 50 60 Days FIG. 12. Specificity (^) of the maintenance antigen. Tolerance persisted only in mice given SRBC every 5 days. *, No (^) maintenance; o, HRBC maintenance; *, SRBC maintenance.
must be given in a dose or (^) form that does not stimulate antibody synthesis (Dresser (^) &
an immune response in animals not given the drug. Furthermore, unlike conventional
Our (^) explanation of these observations rests on the unproven (^) assumption that differentiat- ing, proliferating, antigen-selected (^) cells are more sensitive to the cytotoxic effects of cyclo-
sensitive cells (DeWys & (^) Kight, 1969), which, on stimulation by antigen, finally die. (^) What is more important is that smaller doses of the drug are (^) effective only after the antigenic
Immunological tolerance in mice 99
SCHWARTZ, R.S. (1968) Immunosuppressive drug therapy.^ Human^ Transplantation, p.^ 440. Grune &^ Stratton, New York. SCHWARTZ, R.S. & DAMESHEK, W. (1959) Drug-induced immunologic tolerance. Nature (Lond.), 183, 1682. SCHWARTZ, R.S. & DAMESHEK, W. (1963) The role of antigen dosage in drug-induced immunologic tolerance. J. Immunol. 90, 703. SEVER, J.L. (1962) Application of^ a^ microtechnique^ to viral^ serological^ investigations.^ J.^ Immunol.^ 88, 320.