J mice during adjuvant induced amyloidogenesis

CELLULAR

IMMUNOLOGY

70, 170- 179 ( 1982)

Natural Cytotoxicity in AKR/ J Mice during Adjuvant Induced Amyloidogenesis’ STEVEN

A. FUHRMAN,’

DAVID AND KEITH

R. PARKINSON,~‘~ SAMUEL D. WAKSAL,’ P. W. J. MCADAM~

Tufts Cancer Research Center, 136 Harrison Avenue, Boston, Massachusetts 02111 Received February 2, 1982; accepted March 30, 1982 The induction of amyloidosis in AKR mice has previously been shown to be associated with a decrease in the incidence of spontaneous thymic leukemia (P. Ebbesen, Bri?. J. Cancer 29, 76, 1974). Amyloid induction with azocasein depressesthe activity of the natural killer (NK) cell, a cell believed to be important in the protection against the development of malignancy. In the present studies, therefore, we examined the response of the NK cell to the induction of amyloidosis in AKR mice. Rapid and long-term depression of NK cell activity against YAC-1 tumor cells was noted following intraperitoneal administration of complete Freund’s adjuvant enriched with Mycobacterium butyricum. Mixing studies suggested that active suppression did not account for the observed decrease in NK cell activity. Although some NK cell activity was noted in ascitic Auid, redirection was not felt to account for the rapid and dramatic reduction in splenic NK cell activity. Furthermore, serum from adjuvant-treated but not control mice was found to significantly inhibit NK cell activity in vitro. These studies therefore suggested a role for a serum factor in the depression of this activity. The apparent paradox of decreased NK cell activity in a setting of diminished leukemogenesis is considered in relation to previous studies describing impaired T-cell functioning after the induction of amloidosis.

INTRODUCTION Spontaneous thymic leukemia develops at high incidence in the inbred AKR mouse strain. Advances in the understanding of the pathogenesis of this have been recently reviewed (2, 3). Ebbesen (1) made the initial observation of a significant decrease in the incidence of spontaneous thymic leukemia in AKR mice after an amyloidosis-inducing protocol. The incidence of leukemia dropped from 82 to 32% when 2-month-old female AKR mice were given 30 consecutive daily injections of casein. This regimen was further associated with a 55% incidence of amyloidosis, ’ Supported by a grant from the Cancer Research Institute, Inc., Program Project CA24530, and Contract N-OICB-74 150, as well as a grant from National Institutes of Arthritis & Metabolic Diseases, AM26501. This work was presented in part at the National Student Research Forum, Galveston, Texas, April 23-25, 1980. ’ Recipient of an NIH Student Research Fellowship Award. ’ Fellow of the Medical Foundation, Inc. 4 Author to whom reprint requests should be addressed. ’ Scholar of the Leukemia Society of America. 6 Senior Investigator of the Arthritis Foundation. 170 0008-8749/82/090170-10$02.00/0 Copyright 0 1982 by Academic Peers, Inc. All rights of reproduction in any form reserved.

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while the coexistence of leukemia and amyloidosis was less frequent than statistically expected (P < 0.001). Marked immunodysfunction has been associated with the induction of amyloidosis in a number of strains of mice (4-10). Recently, depression of natural cytotoxicity was observed in CBA/J mice following azocasein induction of amyloidosis ( 11). The natural killer (NK) cell is distinct from classic immunologic effector cells and can directly lyse various neoplastic and virus-infected cells in vitro, in the absence of prior sensitization ( 12- 14). Their possible role in a surveillance system against the development of malignancy has been proposed (13). Therefore, Ebbesen’s observation of a decreased incidence of spontaneous malignancy in association with the induction of amyloidosis (1) presented to us an apparent paradox. We therefore initiated an investigation of NK cell biology in amyloid induction in AKR/J mice. Amyloidosis was induced with a single intracomplete Freund’s adjuperitoneal dose of Mycobacterium butyricum-enriched vant (CFA + Mb), a previously described regimen ( 15, 16). The results demonstrate marked alteration in NK cell biology following this protocol. MATERIALS

AND METHODS

Mice. Four- to six-week-old female AKR/J mice were obtained from Jackson Laboratories (Bar Harbor, Maine) and maintained in the Tufts University Animal Facility. Amyloidogenic protocol. The amyloidogenic agent was prepared by thoroughly mixing 1 g Mycobacterium butyricum (Difco Labs) in 10 ml sterile saline with 10 ml of complete Freund’s adjuvant. Animals were treated by intraperitoneal injection of 0.2 ml of the M. butyricum-enriched complete Freund’s adjuvant (CFA + Mb) (15, 16). Control mice were untreated. Spleen cell preparation. The spleens of experimental animals were removed and gently teased between two frosted glass slides in a petri dish containing Hepes (N2-hydroxyethylpiperazine)-buffered (10 mM) Hanks’ balanced salt solution (HBSS). Erythrocytes were lysed by a lo-min, 37°C incubation with Tris-buffered ammonium chloride. The cells were washed with HBSS + 10 mM Hepes, resuspended in RPM1 1640 (Gibco, Grand Island, N.Y.) supplemented with 2-mercaptoethanol (5 x IO-‘), Penicillin G (100 units/ml)/streptomycin (100 mg/ml), nonessential amino acids ( 1% v/v), L-glutamine (2 mM), Hepes ( 10 mM), and 10% heat-inactivated fetal calf serum (FCS). Cell suspensions were then filtered through 40pm-diameter nylon gauze to remove tissue debris. Tumor targets. YAC-1 is an in vitro carried T-lymphoma line which is susceptible to NK cell lysis (17). These target cells were suspended for 1 hr at 37°C in supplemented RPM1 1640 + 15% FCS to facilitate labeling with 300 &/ml sodium chromate (New England Nuclear, Boston, Mass.). After chromium labeling, the cells were washed three times with HBSS + 10 mM Hepes, and resuspended in supplemented RPM1 1640 + 10% FCS at 1.25 X lO’/ml. NK cell assay. Effector spleen cell suspensions were adjusted to a concentration of 1.25 X lO’/ml and serially diluted to yield effecter/target (E/T) ratios of lOO/ 1, 50/l, 25/l, and 12.5/l. Then 0.1 ml effecters and 0.1 ml targets were added to round-bottom microtiter plates (Linbro Scientific, Hamden, Conn.). The plates were centrifuged for 3 min at 60g (Beckman Model TJ-6, Beckman Instruments,

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Anaheim, Calif.) and incubated at 37°C for 4 hr. The plates were then centrifuged at 260g for 10 min and 0.1 ml of the supernatant was removed and counted for gamma radiation (Beckman Instruments). Spontaneous S’Cr release was determined for target cells incubated with supplemented RPM1 1640 + 10% FCS. Release of “Cr was quantitated with respect to the maximum release (freeze-thaw specimens) and expressed as percentage specific lysis. Percentage specific lysis was calculated as CPmexpcrimental - cPmspntaneous deas x 100. Cpmfrceze-thaw

-

cPmspontaneous

relcase

Mice were tested individually; the standard errors (SEM) of quadruplicates from a single mouse were consistently less than 5%. Poly Z:C induction of NK activity. To enhance NK cell activity, 100 pg polyinosinic-polycytidylic acid (PIC, Microbiological Associates, Bethesda, Md.) in 0.1 ml physiologic saline was administered intraperitoneally 24 hr prior to the assay of splenic NK cell activity. This method and timing of administration has been shown to give peak levels of NK cell activity and is the result of interferon production by macrophages (18). Mixing experiments. To test for the presence of active suppressive influences of NK cell activity, mixing experiments were performed. Spleen cells from CFA + Mb-treated AKR/J mice were mixed with spleen cells from control mice known to have spontaneous NK cell activity. Different mixing ratios were used, calculated to a final effector cell concentration of 1.25 X lo7 cells/ml. A constant effector/ target cell ratio of 100/l was therefore maintained, and NK cell activity in the variously mixed populations was compared to that in the unmixed population at the same E/T ratio. Amyloid ident$cation. Splenic fragments were examined histologically for the presence of amyloid using alkaline Congo red staining and polarization microscopy to identify the green birefringence of amyloid deposits. Radioimmunoassay for SAA. Levels of serum amyloid A (SAA) were estimated using a single antibody solid-phase radioimmunoassay. Antibodies were raised in rabbits to alkali-denatured mouse amyloid fibrils isolated from organs of animals with secondary amyloidosis. The antibodies were affinity purified to murine AA protein. The wells of flexible plastic microtiter plates (Scientific Products) were coated with these mouse AA antibodies and spare sites filled with 1% Tween 20, pH 7.8. A standard inhibition curve was generated by incubating for 18 hr at 4°C in triplicate wells with a known amount of AA at the same time as ‘251-labeled AA. The inhibition of maximum binding of ‘251-AA by unknown samples allowed quantitation of AA cross reactivity, expressed as micrograms per milliliter. Samples to be tested by radioimmunoassay were treated with formic acid for 24 hr at 56°C in order to dissociate SAA and allow for reproducible assay of the expressed AA antigenic sites. Mouse serum e#ect on NK cell activity. The NK cell activity of spleen cells from a young, untreated AKR/J was assayed in the presence of various concentrations of mouse serum from either control or experimental mice with known high levels of SAA. Mouse serum was added to effector cell suspensions in complete RPM1 and incubated for 1 hr at 37°C. Serum was diluted with complete RPM1 to yield incubating concentrations of 20, 5, 2, and 1% mouse serum. 5’Cr-Labeled YAC-

NK CELLS

IN AKR/J

NUMBER

DAYS AFTER

AMYLOIDOGENESIS

173

TREATMENT

FIG. 1. Splenic NK cell activity in CFA + Mb treated (m) and control (0) female AKR/J mice. Assay was against YAC-1 at SO/l effecter/target ratio at various times after treatment. CFA + Mb was injected ip at 4-6 weeks of age. Each point represents the mean percentage specific lysis + SEM of three individually assayed mice.

1 tumor targets were then added, halving the serum concentration, and the standard NK cell assay was performed. Serum effect on effector cell viability and spontaneous “Cr release by YAC-1 targets were noted as additional controls. RESULTS Induction of amyloidosis. Amyloid induction, by the method described by Ram et al. (15) caused hepatic and splenic deposition of amyloid in treated mice within 2 weeks of treatment. No amyloid deposition occurred in control animals. NK cell activity in preamyloidotic and amyloidotic mice. Spontaneous NK cell activity against YAC-1 target cells was measured at various times following treatment with the amyloid-inducing regimen. Within 3 days of treatment, the spontaneous splenic NK cell activity was virtually abolished and remained low throughout the 9 weeks of testing (Fig. 1). NK cell activity in lymph nodes and bone marrow (not shown) was similarly depressed. By 12-15 days after treatment marked ascites had developed in the CFA + Mbtreated mice. To determine whether redistribution of NK cells to the peritoneal cavity was a partial explanation for the long-term diminution of natural cytotoxicity in lymphoid tissues, NK cell activity of cells obtained from ascitic fluid was measured. The mononuclear population was found to effect 9.3 f 0.7% (SEM) specific lysis at 50/l E/T 3 weeks after treatment, when 1.2 + 0.7% lysis was produced by spleen cells. Treatment with an amyloid-inducing regimen was therefore associated with a rapid loss of splenic natural cytotoxicity against YAC- 1. Furthermore, no spontaneous NK cell activity was seen throughout the 9 weeks of observation in lymphoid tissues. Mixing experiments. We next determined whether the loss of NK cell activity following CFA + Mb treatment was due to a suppressor population. Control spleen cells with normal levels of NK cell activity and treated splenocytes were mixed in

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various ratios (Fig. 2). An effect characteristic of dilution of NK cell activity was found, but there was no evidence for suppression. Thus lysis by a 100/l E/T ratio using a 1:1 mixture of control and treated cells was not significantly different from that seenwith a 50/ 1 E/T ratio, which represented an equivalent number of normal cells. The results suggest that the low NK cell activity is, therefore, more likely due to the elimination of cells with NK cell activity by CFA + Mb treatment, than to suppression of the NK cells by another population. Response to interferon induction. Intraperitoneal injection of the synthetic RNA PIC has previously been shown to enhance splenic NK cell activity through interferon production (18). In older mice, with low spontaneous NK cell activity, interferon inducers are able to stimulate significant natural cytotoxicity ( 19). A similar effect has been documented in two other experimental systems with absent spontaneous NK cell activity. In both the split-dose radiation C57B1/6 radiation leukemia system (20) and the NK cell impaired genetic variant C57B1/6 beige (bg’/bg’) mouse (21), interferon has been found to induce NK cell activity, albeit lower levels. Intraperitoneal injection of PIC in CFA + Mb treated AKR/J mice 24 hr before assay produced a partial restoration of splenic NK cell activity (Table 1). Thus, the interferon inducer PIC restores NK cell activity, though not to stimulated control levels, similar to the data from other models with reduced NK cell activity. SAA response. The amino terminal portion of serum amyloid A (SAA), possesses amino acid sequence identity with protein AA, the major amyloid fibril protein found in animals with secondary and experimental amyloidosis (22, 23). SAA has further been identified as a potent immunoregulator (24). To correlate SAA levels with both the marked NK cell depression and the expected amyloidotic changes, serum samples were obtained from mice at the time of each assay. Serum levels of SAA, as measured by RIA, peaked 9 days after treatment at 421 + 11.5 gg/

CFA+Mb

, 2/l

l/l

l/2

,

CONTROL

CFA + iib/CONTROL

FIG. 2. NK cell activity after mixing control and treated (CFA + Mb) splenic populations. Treated cells were from 6-week-old female AKR/J, 14 days after ip injection of CFA + Mb. Percentage specific lysis at 100/l effecter/target ratio (0) against YAC-1 tumor targets. 50/l effecter/target ratio (0) noted as an equivalent. Vertical bars represent the standard error of the mean in the assay.

175

NK CELLS IN AKR/J AMYLOIDOGENESIS TABLE 1 PIC Effect” on CFA + Mb-Treated and Control AKR/J Splenic NK Cell Activityb Percentage specific lysis ET Control Control + PIC CFA + Mb CFA + Mb + PIC

100/l 13.4 f 23.7 + 3.3 f 15.2 f

50/l 2.4 8.8 1.6 4.3

10.2 + 14.6 + 3.1 + 10.3 f

1.2 4.9 0.5 3.5

a 100 rg polyinosinic-polycytidylic acid (PIG) in 0.1 ml normal saline injected intraperitoneally 24 hr before NK cell assay. Control mice received 0.1 ml normal saline IP. b 5’Cr release assay with YAC-1 target cells. Effecter/target ratio as noted, exposure time 4 hr at 37°C. Values represent percentage specific lysis + SEM.

ml (SEM) (control 1.5 pg/ml), and were still elevated 9 weeks after treatment (Fig. 3). At that time specific lysis by treated spleen cells was 1.8 + 0.3%, whereas control spleen cells produced 17.1 + 2.8% lysis of YAC-1 targets (100/l E/T); therefore, NK cell activity was depressed throughout the period of SAA elevation. NK cell activity did not, however, increase as serum SAA concentrations fell. Mouse serum efect on NK cell activity. Table 2 contains the results from a representative experiment illustrating the effect of different concentrations of serum from treated or control mice on normal splenic NK cell activity. The results show that serum from CFA + Mb-treated animals can directly inhibit NK cell lysis of YAC-1 targets. Appropriate controls showed that this effect was not due to alteration of the viability of effector cells, or to increased spontaneous release of chromium from labeled YAC- 1 targets. These results are consistent with a direct serummediated effect on NK cell activity.

NUMBER DAYS AFTER TREATMENT

FIG. 3. Concentration of serum amyloid A (SAA) in female AKR/J mice at various times after CFA + Mb treatment of 4- to 6-week-old animals. Results represent the concentration of SAA (@g/ml) in pooled sera from three individual mice + SEM of the assay. Control values were considerably less than 1.5 rg/ml.

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TABLE 2 Serum Effect on AKR Splenic NK Cell Activity” Percentage specific lysis of YAC-I Final serum concentrationb

E:T

100/l

50/l

10% c 2.5% C 1% c 0.5% c

14.0 + 1.2 15.7 -c 2.9 17.0 * 2.1 16.2 k 1.4

10% CFA + Mb 2.5% CFA f Mb 1% CFA + Mb 0.5% CFA + Mb

1.9 f 9.9 f 13.2 + 15.7 f

1.8 1.5 1.4 1.7

25/l

11.3 iz 9.9 k 10.7 + 8.2 +

1.6 1.1 1.9 1.8

7.4 + 6.5 + 7.5 + 6.4 +

1.1 1.6 3.1 1.4

3.3 + 8.0 f 6.9 k 9.6 k

1.0 1.9 2.0 2.0

2.2 + 4.9 + 5.0 + 3.9 k

1.4 1.5 1.2 2.3

’ “Cr release assay with YAC-1 target cells. Effecter/target ratios as noted. Incubation time 4 hr at 37°C. Specific lysis of YAC-1 targets is reported +SEM. b Splenic effecters from 6-week-old female AKR/J mice were incubated for 1 hr at 37°C with noted serum in 100 ~1. YAC-1 targets in 100 pl were then added to yield final serum concentration. ’ Serum from AKR/J mice 13 days after intraperitoneal injection of CFA + Mb (maximum SAA concentration during incubation, that is, with 20% serum, was 60 fig/ml).

DISCUSSION Intraperitoneal injection of enriched-complete Freund’s adjuvant into AKR/J mice, a protocol previously demonstrated to be amyloidogenic ( 15, 16), resulted in amyloidosis and was associated with both rapid and long-term depression of NK cell activity in AKR lymphoid tissues. The experimental induction of amyloidosis in AKR mice has previously been shown to decrease the high spontaneous incidence of leukemia normally associated with this strain (1). After 30 consecutive daily casein injections in female AKR mice, Ebbesen revealed a 55% incidence of gross amyloidosis associated with a drop in the incidence of leukemia from 82 to 32% (1). Several investigators have revealed marked alterations in lymphoid function during amyloid induction (4-10). The histologic observation of depletion of thymicdependent splenic tissue following the induction of amyloidosis (25) was corroborated by Hardt and Claesson’s (26) finding of a progressive decrease in the number of Thy-l-bearing lymphocytes in mouse spleens following casein treatment. In grossly amyloidotic mice, T-cell function was impaired as evidenced by poor reactivity to T-cell mitogens (8, 10, 27), delayed homograft rejection (7), and inability to induce normal graft-versus-host reaction (5). In preamyloidotic mice, spleen cells failed to generate cytotoxic responseswhether sensitized to parental determinants, trinitrophenol-modified syngeneic cells, or allogeneic cells (6). In addition, histologic studies have shown amyloidogenic protocols to induce marked thymic cortical involution (27). While amyloid induction is associated with marked T-cell impairment, normal or increased B-cell responses have been noted. Scheinberg and Cathcart (9) demonstrated increased B-cell response to mitogens and enhanced primary responses to T-independent antigens. Amyloidogenic regimens are furthermore associated with long-term polyclonal antibody formation (28, 29).

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The natural killer (NK) cell is a bone-marrow derived cell, distinct from classic lymphoid effecters ( 12- 14) which exhibits anti-neoplastic activity (13, 14, 30). The amyloidogenic Freund’s adjuvant protocol caused a marked decrease of NK cell activity against YAC-1 targets in AKR mice, as well as in the low spontaneous leukemia C57B1/6 strain (Fuhrman et al., unpublished data). Depression of natural cytotoxicity was previously reported in CBA mice after azocasein treatment (11). However, strain susceptibility to the development of amyloidosis was recently shown to be independent of spontaneous NK cell activity (3 1). Mixing experiments offered no evidence of active suppression of NK cell activity. These data were therefore most consistent with the elimination or alteration of cells bearing NK cell activity. The appearance, after 2 weeks, of some NK cell activity in the ascitic fluid of treated mice suggested a possible alternative mechanism; i.e., the redirection of NK cell populations to the peritoneal cavity. However, considering the rapid depression of NK cell activity, the delayed development of ascites, and the relatively few cells present in the peritoneum, we feel that redirection offers only a partial explanation of this adjuvant-induced phenomenon. Furthermore, NK cell activity could not be fully restored using the interferon inducer PIC. Similar partial restoration of low NK cell activity by PIC has been noted in the mutant C57B1/6 beige mice (21), in split-dose irradiated C57B1/6 mice (20) and in strontium-treated mice (32). When splenic effecters were incubated with serum from CFA + Mb-treated mice, a dramatic inhibition of NK cell activity occurred. This effect could not be reproduced with normal mouse serum. Depression of NK cell activity against YAC-1 targets was directly related to the incubating CFA + Mb-induced serum concentration. Immune complexes could not be implicated in this phenomenon as we have previously found no effect of even high concentrations on NK cell activity (Parkinson et al., unpublished data). Adjuvant-induced serum, besides having a rich immunoglobulin fraction (28, 29), was shown to contain high levels of serum amyloid A (SAA), an acute phase reactant sharing partial amino acid sequence identity with the fibrils of secondary amyloidosis (22, 33). Purified SAA, in concentrations comparable to the present study, was previously shown to significantly depress T-cell antigen responses to sheep red blood cells (24). The investigators were able to reverse the observed suppression with addition of a rabbit anti-amyloid A antibody. Similarly, other acute phase reactants, including cY,-acidglycoprotein (34) C-reactive protein (35, 36), and a*-macroglobulin (37), have displayed significant immunoregulatory capability. Indeed B cells and the Ty lymphocyte subpopulation, that population expressing NK cell activity, have recently been shown to bind C-reactive protein (38). As these proteins and their respective specific antibodies become available further elucidation of in vivo and in vitro NK cell response to acute-phase reactants will be possible. The NK cell has received much recent attention as a possible mediator of antineoplastic surveillance (13, 14, 30). Yet in these studies, we have demonstrated long-term depression of natural cytotoxicity in a situation previously associated with a decreased incidence of spontaneous leukemia ( I), an apparent paradox. One interpretation of these findings is that NK cells play no role at all in protection against spontaneous AKR leukemia; and therefore, depression of natural cytotoxicity and the decreased incidence of leukemia are unrelated phenomena in AKR amyloidosis. Alternatively the changes in natural killer cell and other lymphocyte

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functions, documented above, may represent a more generalized aberration of Tlineage differentiation. Others have suggested that the NK cell is a thymic precursor. Abnormalities in T-cell differentiation play a significant role in the pathogenesis of AKR leukemia (2, 3). These changes have been demonstrated by both histologic and immunologic techniques (39). The AKR leukemogenic virus is the result of recombination between ubiquitous ecotropic virus and a xenotropic virus expressed selectively in the preleukemic thymus (40). The expression of xenotropic virus, furthermore, has been shown to be related to pre-T-cell activation and differentiation (41-43). The results of our studies suggest that differentiation abnormalities may be more important than the protective effect of NK cells in AKR leukemogenesis. Future studies should explore the hypothesis that alterations of T-cell differentiation associated with the induction of amyloidosis, are reflected in diminished xenotropic virus expression, decreased recombinant polytropic virus formation, and therefore a reduced incidence of AKR leukemia. ACKNOWLEDGMENTS The authors would like to express their thanks to Rebecca P. Brightman and Kenneth Green for their technical support during these studies, to Charles Ceurvels for preparing the figures and to Norma McNaughton for typing this manuscript.

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30. Kasai, M., DeClerc, J. C., McVay-Boudreau, L., Shen, F. W., and Cantor, H., J. Exp. Med. 149, 1260, 1979. 31. Wohlgethan, J. R., and Cathcart, E. S., J. Immunol. 127, 1003, 1981. 32. Kumar, V., Ben-Ezra, J., Bennett, M., and Sonnenfeld, G., J. Immunol. 123, 1832, 1979. 33. Sipe, J. D., Ignaczak, T. F., Pollock, P. S., and Glenner, G. G., 1. Immunol. 116, 1151, 1976. 34. Chiu, K. M., Mortensen, R. F., Osmand, A. P., and Gewurz, H., Immunology 32, 997, 1977. 35. Mortensen, R. F., Osmand, A. P., and Gewurz, H., J. Exp. Med. 141, 821, 1975. 36. Mortensen, R. F., and Gewurz, H., J. fmmunol. 116, 1244, 1976. 37. Stein-Steilein, J., and Hart, D. A., Fed. Proc. 37, 2042, 1978. 38. Williams, R. C., Ann. N. Y. Acad. Sci., in press. 39. Metcalf, D., J. Nat. Cancer Inst. 37, 425, 1966. 40. Kawashima, K., Ikeda, H., Hartlety, J. W., Stockert, E., Rowe, W. P., and Old, L. J., Proc. Nat. Acad. Sci. USA 73, 4680, 1976. 41. Staber, F. G., Schlaefli, E., and Moroni, C., Nature (London) 275, 669, 1978. 42. Moroni, C., and Schumann, G., Nature (London) 269, 600, 1977. 43. Schumann, G., and Moroni, C., J. Zmmunol. 120, 1913, 1978.