Spontaneous regression of friend virus-induced erythroleukemia IX. Role of complement in leukemia regression

Leukemia Research VoL 6, No. 5, pp. 703-710. 1982. Printed in Great Britain.

0145-2126/82/050703-08S03.00/0 © 1982 Pergamon Press Ltd.

SPONTANEOUS REGRESSION OF FRIEND VIRUS-INDUCED ERYTHROLEUKEMIA IX. R O L E OF COMPLEMENT IN LEUKEMIA REGRESSION CLIFFORD LONGLEY a n d PHILIP FURMANSK! Department of Cell Biology, AMC Cancer Research Center and Hospital, 6401 West Colfax Avenue, Lakewood, CO 80214, U.S.A. and Department of Biology, Michigan Cancer Foundation, 110 East Warren Avenue, Detroit, MI 48201, U.S.A.

(Received 14 January 1982. Accepted 2 June 1982) Al~tract--The role of complement was examined in the immunologic,ally.mediated spontaneous regression of erythroleukemia induced by the RFV strain of the Friend virus complex, Hemolytic complement levels were not significantly altered during the leukemic, regressed and recurrent phases of the disease. No correlation was observed between leukemia regression and complement levels in normal, C5-deficient and hybrid mice. No correlation was observed between serum complement activity and leukemia recurrence or mortality due to leukemia, The data suggest that hemolytic complement, and thus those immune effector functions specifically dependent on full complement activity, are not involved in leukemia regression and do not influence the course of the disease.

Key words: Complement. erythroleukemia, regression.

I NTRODU CTION

CONVENTIONAL strains of the Friend virus complex (FV) induce a progressive, lethal erythroleukemia in susceptible mice. This disease is characterized by massive splenomegaly, immunosuppression, viremia and death 1"5,10]. Our laboratory has reported that a particular variant of the Friend virus complex, RFV, causes an erythroleukemia initially identical to that induced by FV, but which spontaneously regresses in half of the leukemic mice [31, 32]. Regression is characterized by the restoration of normal splenic histology and architecture [35], decreased amounts of infectious virus in the spleen and circulation [31] and increased survival. Regression is dependent on normal thymus [7, 12] and macrophage function 123, 24] and is associated with the appearance of potent virus-neutralizing antibodies [2, 33], leukemia cell-specific cytotoxic antibodies [11, 15], and cell-mediated reactivity against viral antigens 1"17]. We are seeking to determine the specific role in regression and the host control of leukemia of each of the immune effector functions detected in regressed mice. Previous studies of the temporal appearance and consistency of detection of the immunologic activities in leukemic and regressed mice have suggested that cell-mediated reactivity plays a major role in disease regression but that humoral immunity may not [17]. Maintenance of the regressed state, i.e. prevention of disease recurrence, also related to immunologic reactivity, is associated with the development of potent virus neutralizing antibody [15]. Abbreviations: FI/, Friend virus complex composed of defective spleen focus-forming virus and a helper MuLV: RFV. regressing Friend virus complex; CHso, the quantity of complement or test serum necessary to lyse 50°0 of the erythrocytes as measured by release of ~aCr; CVF, cobra venom factor. Correspondence to: P. Furmanski, Department of Cell Biology, AMC Cancer Research Center and Hospital, 6401 West Colfax Avenue. Lakewood, CO 80214. U.S.A. 703

704

CLIFFORD LONGLEYand PHILIP F'URMANSKI

Another approach to the analysis of immune functions in regression consists of determining the role of complement. Complement is required for certain immune effector mechanisms (e.g. antibody-mediated cytotoxicity, complement-dependent neutralization) and not for others (cell-mediated cytotoxicity, virus neutralization). Thus, if an intact complement system were necessary for leukemia regression, we would conclude that complement-dependent immune functions were involved in host control of the disease. The results reported here demonstrate that mice lacking functional hemolytic complement system remain fully capable of causing leukemia regression and we conclude that those immune effector mechanisms which specifically require complement are not the significant factors in regression.

MATERIALS AND METHODS Mice. Random-bred Swiss ICR/Ha mice, inbred N/PLCR. BALB/c and the partially inbred substrains of ICR/Ha Swiss mice, NRC7 and $377, were from colonies maintained at the Michigan Cancer Foundation. Breeding pairs of inbred BALB.B mice were supplied by Dr. Frank Lilly and were subsequently bred in our own colony. SIM and SIM.R mice were obtained from colonies maintained at the Michigan Cancer Foundation derived from breeding stock originally provided by Dr. A. Axelrad. Mouse strains deficient in the filth component of complement, A/J, DBA/2J and BI0D2/OSNJ, were purchased from the Jackson Laboratories. Mice were monitored weekly for leukemia development and regression by spleen palpation, which has been shown to be an accurate indicator of the leukemic status of the animal [2, 30, 31]. The mice were considered leukemic when the spleen weight exceeded 0.5 g and regressed when following the development of leukemia the spleen weight decreased to below 0.5 g. Virus. Stocks of RFV were prepared from cell-free spleen homogenates (20~o w/v in phosphate-buffered saline) from leukemic mice as described [30] and stored in sealed ampules at -70°C. Mice 6-10 weeks of age were inoculated i.p. with 0.5 ml ol PBS containing 100 IDso of virus stock. An IDso dose produces leukemia m 50% of weanling Swiss mice within 25 days. Complement assay. The assay for complement levels in mouse sera was performed as described by Berden et al. [1], with modification. One-tenth ml of blood was obtained from the retro-orbital sinus of the mice. Blood was allowed to clot on the side ofa 6 × 50ram culture tube for 15 min at room temperature and then placed at 4°C for 2 h to allow the clot to retract. The serum obtained was centrifuged at 4 C for l0 rain at 409 and immediately assayed. The assay was performed in a dextrose-gelatin-veronal buffer (DGVB z+) containing 4.6mM barbital, 2.6 mM sodium barbital, 0.13 M NaCl, 0.15 mM CaClz, 0.5 mM MgSO4, 0.6°/,~gelatin and 1% dextrose. Sheep RBC in Alsever's solution (Colorado Serum Co., Denver, Colorado) were washed three times, centrifuged at 1600 0 for 5 min and resuslacnded in DGVB 2+ to a concentration of 2 x l0 s cells/ml. The cells were labelled with 100 ~uCi/ml ~'Cr (Na -5 ~CrO,~, New England Nuclear, Boston, Mass.) for I h at 37°C with periodic mixing. The labelled cells were washed twice with DGVB 2+, resuspended and incubated at 4°C for 0.5 h and washed once more. The labelled cells were resuspended to a concentration of 8 x I07 cell/ml, and sensitized by the addition of an equal volume of DGVB 2+ containing a 1:1000 dilution of rabbit antibodies to sheep hemolysin (Capp¢l Laboratories, Dowingtown, Pennsylvania) and incubation for 10 rain at 37~C with frequent mixing. Serial two-fold dilutions of the test mouse sera were made in DGVB 2 +, starting at 1: 10. Fifty lambda of labelled, sensitized SRBC were added to 50 lambda of diluted test sample. The mixture was incubated at 30°C for 90 rain. The reaction was quenched by the addition of 0.9 ml ice-cold DGVB 2 +. The samples were centrifuged for 5 min at 16000. An aliquot of 0.7 ml of the supernatant was removed and counted in a gamma counter (soluble counts/rain) as was the remaining supernatant and pellet (soluble and pellet counts/rain). Controls run with each assay included heat inactivated normal mouse serum diluted 1:20 and freshly redissolved aliquots of two different pools of normal mouse sera which had been lyophilized and stored at - 70°C. Total releasable counts were determined by the addition of distilled water to the labelled, sensitized cells followed by three cycles of freezing and thawing. Spontaneous release of S'Cr in this assay system was always <2~o and total release was always >95~o. Intraassay variation was _+ 10~ and interassay variation was _+20Yo. The percentage of ~tCr released was calculated according to the following formula:

Soluble counts/min/0.7 ~o Total release = Soluble counts/min + (Soluble and pe let counts/min) x 100. The CHs0 titre (dilution of test serum causing 50% lysis of sensitized RBC) was determined using linear regression analysis of Iogits vs the logarithm of the serum dilution. Data are expressed as CHs0 units/ml of undiluted serum, lnterassay variation was corrected by reference to the two control normal mouse sera. Assays of complement levels were also carried out using sensitized rabbit RBC as targets [37, 38]. No significant differences were detected in CHso titres using either rabbit or sheep RBC.

Role of complement in leukemia regression

705

RESULTS

Leukemia regression and complement levels in inbred mice Mouse strains differ in their ability to cause regression of RFV-induced erythroleukemia. These differences are regulated by host genotype, and probably involve genes of RFV series, two of which map within or adjacent to the H-2 locus [8]. Mouse strains also vary in their levels of serum hemolytic complement activity (and up to 40% of the inbred laboratory strains have no detectable complement activity [3]). Therefore, we examined the relationship between leukemia regression and serum complement activity in a series of mouse strains with different genetically determined capacities to cause regression and different hemolytic complement levels. As shown in Table 1, there was no association between complement activity and regression. Two of the three regressor mouse strains, SIM and the partially inbred regressor Swiss line, $377, had no detectable hemolytic complement activity. In addition to those shown in Table l, the strains DBA/2J and A/J are also regressors and are deficient in complement activity (see below and [6, 29].) Leukemia regression and complement levels in Swiss mice To substantiate the lack of association between complement activity and regression further, studies were carried out using the random-bred Swiss ICR/Ha strain. Assays of serum hemolytic activity in individual Swiss mice revealed that this strain is heterogeneous for expression of complement activity. Of 380 mice tested, 73% had high levels, while the remainder had no detectable activity. Mice randomly selected from the complement expressor and non-expressor groups were inoculated with RFV and observed for leukemia development, regression, recurrence, and death (Table 2). No significant differences were detected between the two groups in any of these disease parameters.

TABLE 1. LEUKEMIA REGRESSION AND COMPLEMENT LEVELS IN INBRED MICE Mouse phenotype

Mouse stTain

90 Leukemia regression*

CHso (units/ml)5" 164 < 1 < 1

Regressor

N/PLCR SIM $377

37 29 100

Progressor

BALB/c BALB/B SIM.R NCR7

0 0 1 0

77 49 147 191~.

*Determined using from 42 to 263 mice/strain. t M e a n , determined from assays of 5-79 mice/ strain. +*Offive mice tested, three had no detectable complement activity and two did. The m e a n is for the two positive mice.

TABLE 2. EFFECT OF COMPLEMENTON THE COURSE OF RFV-INDUCED LEUKEMIA

Complement + -

Number inoculated

Number leukemic

N u m b e r of regressions

N u m b e r of recurrences

N u m b e r of leukemic deaths

47 26

46 24

22 12

13

11

6

7

CLIFFORD LONGLEY and PHILIP F'URMANSKI

706

I 3

o

u

•

v

o

o

•

.I

o

w

• o

~100 .J kz

o o o

w ..J

o

o

o

|

o

8

o o

,

I o

o

o

uo

**** S$$ A 8 C LOW x LOW

0"*

***

A O C LOW x HIGH

A

B

C

HIGH x HIGH

FIG. 1. Complement levels and erythroleukemia in Swiss/ICR hybrid mice. Low and high refer to complement levels in the parental animals. A, Progressor mice; B, regressor mice; C, uninfected mice.

To determine whether small, genetically regulated differences in complement levels might influence the course of the leukemia, mice selected for either high or low complement activities were mated and the progeny tested for complement levels and their ability to cause leukemia regression. The results showed that the progeny of the parental mice which exhibited no complement activity were also devoid of complement but were still capable of causing leukemia regression. Although the proportion of the FI progeny of the low x low mating whose leukemia regressed was low, there was no statistically significant difference in regression among the groups (•2 = 4.49, df = 2, p > 0.i), nor was there a difference between the low complement mice in groups A and B and the high complement mice in groups B and C (X2 = 2.32, df= 3, p > 0.1) (Fig. 1). The distribution of activity in randomly selected Swiss ICR/Ha mice and the mating experiments were compatible with a single gene difference between the ICR/Ha complement expressors and non-expressors, with two alleles at this locus, the one for expression being dominant. (This assumes that the high complement parent of the B mating was heterozygous at this locus, and that the B mating represented a backcross to a homozygous non-expressor parent.) However, further genetic analysis would be required to confirm this hypothesis.

Analysis of the complement deficiency in Swiss ICR/Ha and SIM mice Since a large proportion of inbred mouse strains are known to be deficient in hemolytic complement activity, and this deficiency has been shown to be a consequence of the inability to synthesize a functional C5 component [29], we considered that lack of C5 was also the most likely cause for lack of detectable hemolytic complement in the negative ICR/Ha and SIM mice. This possibility was directly tested by determining whether sera from mice known to lack C5 but to contain all of the other complement components were capable of correcting the deficiency in sera from ICR/Ha or SIM mice.

Role of c o m p l e m e n t in leukemia regression

707

TABLE 3. THE EFFECT OF MIXING O N C H s o TITRES OF VARIOUS MOUSE $ErA

Strain

ICR I+)

ICR (+) ICR ( - ) PLCR SIM SIM.R BIOD2/OSN DBA/2J A/J ANMS*

400 231 411 465 520 143 170 248 100

ICR (-) PLCR

< 1 48 < 1 247 < 1 < 1 < 1 < 1

131 154 147 30 45 119 20

SIM

< 1 425 < 1 < I < 1 < 1

SIM.R

BIOD2/OSN

652 127 129 241 96

< 1 < 1 <1 <1

DBA/2J

< 1 <1 <1

A/J

ANMS*

<1 <1


*Heat-inactivated m o u s e serum.

As shown in Table 3, serum from B10D2/0SN, DBA/2J or A/J mice (which lack C5) when mixed with sera from ICR/Ha or SIM mice did not result in functional hemolytic complement activity. In addition, mixtures of ICR/Ha and SIM sera did not result in complement activity. These results, together with the preliminary genetic analysis (see above), although not conclusive, strongly suggest that all SIM and some Swiss ICR/Ha mice lack complement activity because they are homozygous for the recessive, nonexpressor allele at the genetic locus specifying synthesis (or secretion, see [29]) of the C5 component. Complement consumption in leukemia mice Although our data showed conclusively that mice without detectable hemolytic complement activity could cause leukemia regression, the possibility could not be eliminated that in those regressor animals with a fully functional complement cascade, this immunological mediator did play a role in regression. To assess this possibility, individual Swiss ICR/Ha mice were tested for complement levels, inoculated with virus and retested for complement levels at weekly intervals throughout the course of their leukemia and leukemia regression. The results are summarized in Fig. 2. (For simplicity, results are only shown for the entire population at the indicated test points. The results for individual animals tested weekly are the same.) We found that there was no significant change in complement levels during leukemia development, no difference in complement activity in leukemic regressors and progressors and no detectable complement consumption in animals just prior to or following regression. The mean complement levels in both progressor and leukemic regressor mice at 44 days post-inoculation was decreased slightly from previous samples and from the normal controls, but the difference was not significant and probably reflected the substantial tumor burden and attendant hematological abnormalities in these mice at that time. DISCUSSION Hemolytic complement activity does not appear to play an important role in the spontaneous regression of RFV-induced erytholeukemia. Mice that are genetically deficient in the C5 component of complement are fully competent to cause leukemia regression. Furthermore, regressor and progressor mice do not differ in their initial complement levels or in the levels detected through the course of their diseases. Our previous studies have demonstrated that regression is immunologically mediated. Some leukemic and most regressed mice develop a complex and varied series of immuno-

CLIFFORD LONGLEYand PHILIP FURMANSKI

708

300

o

-==oo

•

.J

•

0

•

-=

-= ~= teoo 3

s T

I'

o

_

. ~** °° o

T -6

8

~

. it

H -

II ~

~

°

~

"o.

o

0o

o

o

• ,

,

** • *8*

* a

o

I, *

o o

**

o

DAYS

tt .

o +o

07

22

o

, g

+

0+ l'l°°:ol °'' 6

..

44

I

70

POST- INOCULATION

FIG. 2. The effect of erythroleukemia on serum CHso titres of Swiss/ICR mice, A. Progressor mice; B, regressor mice; C, uninfected mice. Closed symbolsin group B at 44 days post inoculation represent regressed mice. logic reactions against the virus and leukemia cells. These include specific T-cell reactivity [7, 12] complement-dependent leukemia cell cytotoxic antibody [11, 15], complement-dependent virus-neutralizing antibody and complement-independent virusneutralizing antibody [13, 15]. Studies on the appearance and consistency of these different immunologic activities have indicated that T-cell mediated immunity is central to leukemia regression [17], while high levels of neutralizing antibody are associated with the prevention of leukemia recurrence in regressed mice [15]. The results presented here support this contention since these activities are independent of hemolytic complement, t h e other immunologic effectors detectable in regressed or leukemic regressor mice, which require complement for activity, likely play little or no role in regression. Studies of the effects of complement depletion on host defenses against virus infections have been carried out by other investigators. Hicks et al. [14], reported that de-complemented or C5-deficient mice infected with influenza virus experienced prolonged infection and higher morbidity and mortality than did complement sufficient animals. Hirsch et al. [16], reported that the course of Sindbis virus infection was altered by complement depletion of BALB/c mice. In these studies complement was shown to play a role in both effective host resistance against Sindbis virus as well as in the immunopathologic consequences of infection. In related studies, Drake et al. [9] and Marquez et al. [25], showed that complement was depleted in tumor-bearing mice and hamsters, respectively. Depletion was the result of the presence in tumor-bearing animals of increased amounts of immune complexes. Kassel et al. [19], reported that infusion of normal sera into AKR leukemic mice caused a marked reduction in the size of the affected lymph nodes and spleens. The effective component in these sera was apparently C5. Thus, in this system, induced

Role of complement in leukemia regression

709

leukemia 'regression' required an intact complement cascade and was likely a function of complement-dependent immunologic effector mechanisms such as antibody-mediated cytotoxicity. Differences between the AKR and RFV diseases, including the target cells (lymphoid vs erythroid), latent period (long vs short), type of initiating virus (endogenous vs exogenous) or mode of transmission (vertical transmission vs exogenous inoculation of adult mice) could account for the differences in the role of complement. In some studies of the role of complement in resistance to infectious diseases, cobra venom factor (CVF) was used to deplete complement, based on its ability to activate the cascade at the level of C3. However, the impurity of available CVF preparations, the transience of its effects and the formation of anti-CVF antibodies by multiple inoculation, mitigated against its use in our experiments. The studies reported here were based on the inability of certain mouse strains to complete the hemolytic complement cascade due to a genetic deficiency in component C5. It remains possible, however, that complement components other than C5 might play a role in leukemic regression. For example, virus sensitized with antibody can be neutralized (or cross-linked) using only components C1-C3, a mechanism which could occur in C5-deficient animals [4, 21, 22, 28]. Reactions of the first four components of complement with antigen-antibody complexes form potent mediators, most notably the cleavage products of C3, which may promote phagocytosis [18, 26], stimulate chemotaxis [27], enhance antibody-dependent cellular cytotoxicity or stimulate production of lymphokines by B cells [20, 34, 36]. However, these complement-mediated effects have not been shown to be important effector mechanisms in the host response against ieukemias or oncornaviruses and thus we consider it unlikely that components of the complement system present in C5 deficient mice are involved in regression. We conclude that neither complement nor complement-dependent functions are required for the immunologically-mediated spontaneous regression of RFV-induced erythroleukemia. The immunological responses which appear to be involved in host control of this leukemia are the complement-independent functions, cell-mediated immunity (T-cell reactivity against viral antigens) and v.irus-neutralizing antibody. Acknowledoements--This study was supported by grant CA-14100 from the National Cancer Institute and by an institutional grant from the United Foundation of Greater Detroit.

REFERENCES 1. BERDEN J. H. M., HAGEMAN J. F. H. M. & KOENE'R. A. P. (19781 A sensitive haemolytic assay of mouse complement. J. Immun. Meth. 23, 149. 2. CERNV J., ESSEX M., RICh M. A. & HARDY W. D. (1975) Expression of virus associated antigens and immune cell functions during spontaneous regression of Friend viral murine leukemia. Int. J. Cancer. 15, 351. 3. CINADER B.. DUalSKI S. & W^RDLAW A. C. 0964) Distribution, inheritance, and properties of an antigen, MUBI. and its relation to hemolytic complement. J. exp. Med. 120, 897. 4. DANIELSC. A.. BORSO8T. & RApp H. J. (19691 Neutralization of sensitized virus by the fourth component of complement. Science 165, 508. 5. DENT P. (19721 Immunodepression by oncogenic viruses. Prog. reed, Virol. 14, 1. 6. DIETZ M. & RICH M. A. (1972) Effect of host strain and H-2 type on spontaneous regression of murine leukemia. Int. J. Cancer 10, 99. 7. DlETZ M., FURSlANSKI P.. CLVMER R. & R I c n M. A. (1976) Effect of thymectomy and antithymocyte serum on spontaneous regression of Friend virus induced erythroleukemia. J. hath. Cancer Inst. 57, 91. 8. DIETZ M., FOUCHEY S. & FURMANSKI P. (1981) Spontaneous regression of Friend-virus-induced erythroleukemia. VII. The genetic control of regression. Int. J. Cancer 27, 341. 9 DRAKE W. P., U~GARO P. C. & MARDINEY M. R. (1973) The measurement and manipulation of hemolytic complement levels in tumor-bearing C57BL/6 mice. Biomedicine 18, 284. 10. FglEND C. (1957) Cell-free transmission of a disease having the character of a leukemia. J. exp. Med. 105, 307.

CLIFFORD LONGLEYand PHILIP FURMANSKI

710

11. FURMANSKIP., GOODENOW R., BALDWINJ., CLYMER R. & RICH M. A. (1975) Immune status of mice in which leukemia has spontaneously regressed. Fedn Proc. 34, 973. 12. FURMANSKIP., DIKrz M., FOUCHEYS., HALL L., CLYMERR. & RICH M. A. (1979) Spontaneous regression of Friend murine leukemia virus induced erythroleukemia. IV. Effects of radiation and athymia on leukemia regression in mice. J. natn. Cancer Inst. 63, 449. 13. FURMANSKIP., LONGLEYC., BOLLF_.$C. S., HINES D. L. & DIETZ M. (1980) Spontaneous regression of Friend virus-induced erythroleukemia. VI. Structural and antigenic differences between regressing and conventional strains of virus. J. Virol. 33, 1083. 14. HICKSJ. T., ENNLSF. A., KIM E. & VERBONrrz M. (1978) The importance of an intact complement pathway in recovery from a primary viral infection: influenza in decomplemented and in C5-deficient mice. J. lmmun. 121, 1437. 15. HINES D. L., DIETZ M., MARCELLETTIJ. & FURMANSKIP. (1981) Spontaneous regression of Friend virus induced erythroleukemia. VIII. Humoral immune reactivity in regressed and leukemic mice. Leukemia Res.

5,1. 16. HIRSCHR. L., GRIFFIND. E. & WINKELSTEINJ. A. (1978) The effect of complement depletion on the course of Sindbis virus infection in mice. J. lmmun. 121, 1276. 17. JOHNSONC. S., FOUCHEYS. P. & FURMANSKIP. (1980) Spontaneous regression of Friend murine leukemia virus-induced erythroleukemia. V. Cell-mediated antivirus immunity in mice undergoing erythroleukemia regression and in leukemic mice. J. natn. Cancer Inst. 64, 645. 18. JOHNSONR. B. & STROUD R. M. (1977) Complement and host defense against infection. J. Pediat. 90, 169. 19. KASSELR. L, OLD L. J, CARSWELLE. A., FIORE N. C. & HARDY W. D. (1973) Serum-mediated leukemia cell destruction in AKR mice: role of complement in the phenomenon. J. exp. Med. 138, 935. 20. KESSLER,H. B. & LOBUOLIOA. F. (1980) Interaction of monocytes with tumor cells coated with complement with or without antibody. Cell lmmun. 49, 352. 21. LEDDYJ. P., SIMONSR. L. & DOUGLASR. G. (1977) Effect of selective complement deficiency on the rate of neutralization of enveloped viruses by human serum. J. Immun. !18, 28. 22. LINSCOTTW. D. & LEVINSONW. G. (1969) Complement components required for virus neutralization by early immunogiobulin antibody. Proc. ham. Acad. Sci. U.S.A. 6,1, 520. 23. MARC~LLETTIJ. & FURMANSKIP. (1978) Spontaneous regression of Friend virus-induced erythroleukemia. III. The role of macrophages in regression. J. lmmun. 120, 1. 24. MARCELLETrlJ. & FURMANSKIP. (1979) Infection of macrophages with Friend virus: relationship to the spontaneous regression of viral erythroleukemia. Cell 16, 649. 2S. MARQUEZE. D., BACKENSTOSEJ. M. & CHAPMANJ. L. (1980) Depletion of total hemolytic complement in sera from hamsters bearing herpes simplex type 2-induced tumors. Cancer Res. 40, 713. 26. McCALL C. E. (1979) Complement. S. Med. J. 72, 47. 27. MULLER-EBEgHARDH. J. (1975) Complement. A. Rev. Biochem. 44, 697. 28. OLOSrONE M. B., COOPER N. R. & LARSOND. L. (1974) Formation and biologic role of polyoma virusantibody complexes: a critical role for complement. J. exp. Med. 140, 549. 29. Ool Y. M. & COLTENH. R. (1979) Genetic defect in secretion of complement C5 in mice. Nature, Lond. 282, 207. 30. RICH M. A., GELDNERJ. & MEYERSP. (1965) Studies on murine leukemia. J. hath. Cancer Inst. 35, 523. 31. RIcH M. A., SIEGLER R., KARL S. & CLYMER R. (1969) Spontaneous regression of virus-induced murine leukemia. I. Host-virus system. J. hath. Cancer Inst. 42, 559. 32. RICH M. A., CLYMER R. & KARL S. (1969) Spontaneous regression of virus-induced murine leukemia. II. Influences of environmental factors. J. natn. Cancer Inst. 42, 571. 33. RICH M. A. & CLYMERR. (1971) Host r.esponse in spontaneous regression of murine leukemia. Cancer Res. 31, 803. 34. ROUSEB. T., GREWALA. S. & BABIUKL. A. (1977) Complement enhances antiviral antibody-dependent cell cytotoxicity. Nature, Lond. 266, 456. 35. Russo I., Russo J., BALDWINJ. & RICH M. A. (1976) Histopathology of leukemia regression in virusinduced murine leukemia. Am. J. Path. 85, 73. 36. SANDBERGA. L., WAHL S. M. & MERGENHAOENS. E. (1975) Lymphokine production by C3b-stimulated B cells. J. immun. 115, 139. 37. VAN DUK H., RADEMAKERP. M. & WILLEgS J. M. N. (1980a) Determination of alternative pathway of complement activity in mouse serum using rabbit erythrocytes. J. Immun. Meth. 36, 29. 38. VAN DIJK H., RAOEMAKERP, M. & WILLERSJ. M. N. (1980b) Estimation of classical pathway of mouse complement activity by the use of sensitized rabbit erythrocytes. J. lmmun. Meth. 39, 257.