inhibition of cell-mediated cytotoxicity by corynebacterium parvum

Cancer Immunol. Immunother. 8, 39-44 (1980) ancer LI immunolggy and lJ!mmunotherapy © Springer-Verlag 1980

Inhibition of Cell-mediated Cytotoxicity by Corynebacterium parvum Influence of Dose and Route of Administration

A. Lichtenstein, R. Murahata, and J. Zighelboim

Department of Microbiology and Immunology, UCLA School of Medicine, Los Angeles, California, 90024, USA

Summary. We investigated the effect of altering dose and route of Corynebacterium parvum (C. parvum) administration on the adjuvant's inhibition of cell-mediated cytotoxicity (CMC). Primary in vivo and secondary in vitro CMC of C57B1/6 mice alloimmunized to P815 were depressed if C. parvum was administered systemically (IV or IP) but not when it was given SC. Similarly, only systemic C. parvum generated cells capable of suppressing in vitro CMC. Primary and secondary CMC in spleen was equally inhibited by 700 and 70 ~g, whereas suppressor cell activity was marked with 700 ~g and minimal with 70 ~g. Administration of C. parvum SC admixed with alloantigen resulted in early enhancement and late depression of primary CMC. Secondary CMC was depressed but suppressor activity was absent. Dissociation of CMC depression from suppressor cell generation in- dicates that these phenomena can be separated under certain conditions.

Introduction

Corynebacterium parvum (C. parvum) has varied effects on the cellular components of the immune system. While it potentiates B cell functions, increasing antibody responses to T-dependent and T-independent antigens [5], its effects on T cells are primarily suppressive, causing inhibition of graft-versus-host reactivity [4], delayed cutaneous hypersensitivity reactions [ 15], and proliferative responses to alloantigens and polyclonal mitogens [ 12]. The mechanisms responsible for either its adjuvancy or its immunosuppressive effects are essentially unknown, although it has been suggested that C. parvum-activated macrophages mediate both effects [6, 13].

We have recently described an inhibitory influence of C. parvum upon cell-mediated cytotoxicity (CMC) [9, 10]. When administered IV to mice during an ongoing response to alloantigen, C.parvum depresses primary and

Reprint requests should be addressed to: A. Lichtenstein

secondary CMC and causes the appearance of adherent phagocytic macrophage-like cells that inhibit secondary cytotoxic response of restimulated alloimmune spleen cells. These immunosuppressive effects on CMC may, in part, explain the afore-mentioned inability of C. parvum- treated animals to develop graft-versus-host reactivity and cutaneous delayed hypersensitivity.

C.parvum is an effective immunotherapeutic agent in a variety of syngeneic tumor systems, and therefore the possibility of selective modulation of its effects on cytotoxic T cell functions would be advantageous. The present study was designed to examine whether altering the dose and route of C. parvum administration might mod- ulate its regulatory effects on the expression of cell- mediated cytotoxicity. We reasoned that if the individual effects on CMC depression and suppressor cell generation could be separated by varying dose and route, this would suggest that they are independent phenomena caused by different components of the organism or due to triggering of different cell populations. The results of the study clearly indicate that the inhibitory effects of C.parvum on CMC are dose- and route-dependent. With 70 ~g C.parvum, primary and secondary CMC were markedly inhibited, while generation of suppressor cell activity was only present at low effector-to-target cell ratios. Further- more, administration of C. parvum admixed with alloantigen caused depression of CMC without generation of suppressor cells. The two effects were clearly independent of each other when C. parvum was used under these conditions. We were unsuccessful, however, in defining a dose or route at which the effects on primary and secondary CMC were effectively separated.

Materials and Methods

Animals

Male C57BL/6 (Strong, San Diego, Ca.) mice were used in all experiments. Mice were obtained at 6-8 weeks of age and rested at least 1 week before the initiation of an experiment.

0340-7004/80/0008/0039/$ 01.20

40 A. Lichtenstein et ai.:

A lloimmunization

The P815 mastocytoma was maintained in ascities form by weekly passage in DBA/2 mice, the strain of origin. C57BL/6 mice were alloimmunized by an SC or IP injection of 2 x 107 P815. The SC immunizations were in the right flank. Mice were then divided into two groups. One received IV, IP, or SC (left flank) injections of varying doses of C. parvum (lot 102-V, Burroughs Wellcome, Research Trian- gle Park, NC) 9 days after primary immunization, In some experiments, 700 ~g C.parvumwas admixed thoroughly with 2 x 107 P815 and injected SC in the right flank. The other group served as the immune controls. Five days after C. parvum administration (14 days after alloimmunization) mice were sacrificed for sera collection, assay of primary cell-mediated eytotoxicity, and initiation of restimulated secondary cultures.

Assay of CMC

Mice were sacrificed by cervical dislocation. Peritoneal lymphocytes (PLs) were obtained by lavaging the peritoneal cavity with 10 ml cold minimal essential medium (MEM) (Microbiological Associates, Wal- kersville, MD) containing 5 IU heparin/ml. The spleen and mesenterie nodes were removed aseptically and single cell suspensions prepared by expression of the organs through a stainless steel mesh. Cytotoxic activity was measured, the immunizing cell being used as the target cell in a 51-chromium release assay described by Brunner et al. [1]. Mix- tures of effector and target cells at several ratios were incubated for 4 h at 37 ° C before centrifugation. One half of the supernatant was removed and both samples were counted.

Specific release was calculated according to the following for- mula:

% specific release =

% release experimental group - % background release x 100.

% maximal release

Maximal release was determined by addition of HCL to a final concentration of approximately 1 M, final figures of between 80% and 90% oftotalincorporated counts being recorded. Medium alone served to determine background release, which was 7%-15% of total incorporated counts. Total incorporated counts usually ranged from 3,000--7,000 CPM. The results were fitted by linear regression to a plot of log (E : T) versus % specific release. One lytie unit was defined as the number of effector cells required to cause a specific release of 25%,

Inhibition of Cell-mediated Immunity by Corynebacterium parvum

In Vitro Restimulation

Restimulation of spleen cells in vitro was accomplished by incubating 5 x 106 spleen cells with 5 x 105 mitomycin-C-treated P815 at 37 ° C for 5 days in 2.5 ml MEM supplemented with 10% heat-inactivated fetal calf serum, 5 x 10 -5 M 2-mercaptoethanol, L-glutamine, and antibi- otics. The cells remaining in culture were harvested and tested for cytotoxic activity. Suppressor cell activity was assessed by co-cul- turing spleen ceils from alloimmune animals with spleen cells from C. parvum-treated ailoimmune animals at a 1 : 1 ratio. Cells remaining in culture after 5 days were then harvested and tested for cytotoxicity. Controls consisted of alloimmune spleen cells co-cultured with naive spleen cells and restimulated with an appropriate target. This group al- ways demonstrated cytotoxicity identical with that of restimulated alloimmune cells cultured alone, and thus only the latter control cultures are presented in Tables 2, 4, and 6. Viable cell recoveries after 5 days generally paralleled the degree of cytotoxicity generated in these cultures.

Assay of Cytotoxie Antibody

Mice were bled from the jugular. Blood was kept overnight at 4 ° C before centrifugation to separate serum from the clot. Sera were de- complemented by heating to 56 ° C for 30 min. We then added 0.1 ml 5~chromium-labeled target cells (106/ml) to 0.1-ml aliquots of serial two-fold dilutions of serum and incubated the mixtures at 37 ° C for 30 min. Normal rabbit serum (0.1 ml) was added as a source of complement, and the incubation was continued for a further 30 rain. The reaction was stopped by the addition of 2 ml cold medium and the mixtures centrifuged. One milliliter of supernatant was removed, both samples were counted, and the percentage release was calculated. The titer of the serum was defined as the reciprocal of the highest dilution of serum resulting in a release of over 10% above complement con- trois.

Results

Effects of Route of C. parvum Administration on CMC Response Following SC Alloimmunization

To inves t iga te the effects o f rou te o f C. parvum admin-

i s t ra t ion on the ce l l -media ted i m m u n e re sponse , mice

were i m m u n i z e d SC and then in jec ted on day 9 wi th

Table 1. Effect of varying route of C. parvum injection on primary CMC

C. parvum (CP) route a Spleen CMC Inhibition Nodal CMC Inhibition PL CMC E:T = 100:1 (%) E:T = 100:1 (%) E:T = 50:1 R -+ SD c J~ + SD X _+ SD

inhibition (%)

No CP 35+ 17 -- 41_+ 11 - 51+_9 - IV 12_+ 2 u 66 36_+ 8 <20 28-+8 b 46 IP 8+ 1 b 78 37_+ 5 <20 4 5 + 7 <20 SC 32_+ 5 9 34+ 3 <20 47-+ 9 <20

"In all groups, CP (700 vg) was administered on day 9 after alloimmunization; assay was performed on day 14 b Significantly different (P < 0.05) as compared to corresponding alloimmune control c Mean + SD of four separate experiments

A. Lichtenstein et al.: Inhibition of Cell-mediated Immunity by Corynebaeterium parvum 41

700 ~g C. parvum IV, IP, or SC (Table 1). As shown, comparable suppression of primary CMC in spleen was achieved when C.parvum was given IV or IP. C.parvum given SC had no effect. The cytotoxic capacity of PLs was only inhibited when C. parvum was given IV whereas mesenteric lymph-node responses were totally unaffected regardless of route.

C.parvum administered IV and IP markedly inhibited expression of secondary cytotoxicity in vitro and effectively induced the generation of suppressor cells. By contrast, SC C. parvum had no effect (Table 2).

Humoral immunity was not affected when 700 vg C. parvum was given by any of the routes tested. Serum titers were consistently between 1//8 and ~/~6 for control and all C. parvum-injected groups.

Effects of Varying Dose of I V C. parvum on CMC Following SC Immunization

The effects of IV C. parvum on CMC and generation of suppressor cells are dose-dependent (Table 3). As shown, only 700-¢g and 70-~xg doses of C. parvum were capable of inhibiting primary CMC in spleen. The degree of inhibition was comparable at these two doses. Similarly, these were the only two doses that caused splenomegaly. Conversely, primary cytotoxicity in lymph nodes was comparable to alloimmune controls at all doses tested (data not shown).

Dose dependency was equally observed for the in vitro expression of secondary cytotoxicity and suppressor cell activity (Table 4). As demonstrated, 700 ~g induced the most profound inhibition of secondary CMC and the highest level of suppressor activity. A dose of 70 ~g caused moderate, albeit significant, CMC depression (P < 0.05). Suppressor cell generation after 70 ~g was

only present at lower effector-to-target cell ratios. When these results are expressed as lytic units/106 cells, it is evident that suppressor cell activity generated by 70 ~g was significant, but less impressive than that generated by 700 ~g.

A similar dose dependency was found after C.parvum was given IP 9 days after alloimmunization. Primary cytotoxicity in spleen is inhibited to comparable extents by 700 and 70 ~g, while inhibition of secondary cytotoxicity and generation of suppressor cells was clearly more pro- nounced after 700 ~g.

Specific humoral immune responses of mice treated with varying doses of IV C. parvum were only mildly depressed by 7 9g C. parvum, whereas higher or lower doses caused no significant changes (Table 5).

Intraperitoneal C.parvum was unable to mediate suppression of PL cytotoxicity in mice receiving SC alloimmunization (Table 1). We considered the possibility that alloantigen and C. parvum, or alloantigen-reactive cells and C.parvum had to be in close contact at a site of the immune response to induce its inhibitory effects. To test this, C. parvum was administered IP to animals who had received IP alloantigen 9 days previously. The results, however, are similar to those obtained following SC alloimmunization, i.e., primary CMC was depressed in spleen while remaining normal in PLs.

Subcutaneous Admixture

When SC C. parvum was administered admixed with alloantigen, a biphasic effect was noted. Primary cytotoxicity in spleen and lymph nodes was enhanced (compared with controls) on day 9 after alloimmunization. By day 14, however, mice immunized with alloantigen admixed with C. parvum had depressed primary CMC in

Table 2. Effects of varying route of C. parvum injection on secondary CMC and generation of suppressor cell activity

Source of spleen cells a in CMC Inhibition restimulated cultures E: T = 5 : 1 (%)

+ SD a

1. Alloimmune control, no CP 72 _+ 20 - 2. Mice receiving CP IV 8 _+ 3 e 89 3. Mice receiving CP IP 13 _+ 3 c 82 4. Mice receiving CP SC 70 _+ 6 < 20

Co-culture of 1 + 2 b 24 _+ 5 c 67 Co-culture of 1 + 3 41 _+ 15 c 44 Co-culture of 1+4 73_+ 5 <20

a CP (700 ~g) was administered by different routes on day 9; cells for assay were obtained on day 14 b Cells were co-cultured at a 1:1 ratio c Significantly different (P < 0.05) from alloimmune control

Mean + SD of four separate experiments

Table 3. Effect of dose on C. parvum-induced inhibition of primary CMC a

C.parvum Cells/spleen Splenic CMC Suppression dose X _+ SD b (%)

No CP l l 0 x 106 35 _+ 17 -- CP 700 ~g 169 × 106 12 + 2 c 66 CP 70 ~g 157× 106 6.5+ 6 e 82 CP 7 ~g 120x 106 23 _+20 35 CP0.7 Fxg 116x 106 31 +22 <20

a CP administered IV on day 9; assay on day 14 b Mean + SD of six separate experiments c Significantly different (P < 0.05) from aUoimmune control

42 A. Lichtenstein et al.: Inhibition of Cell-mediated Immunity by Corynebacterium parvum

Table 4. Effect of dose on C. parvum-induced inhibition of secondary CMC and suppressor cell generation a

Source of spleen cells CMC J~ ± SD a Lytic units per in restimulated cultures 10 6 cells

E:T = 25:1 E : T = 5:1 E : T = 1:1

1. Alloimmune control, no. CP 84 ± 11 68 ± 23 36 ± 19 41 ± 15 2. Mice receiving 700 ~g CP 8 ± 4 e 2 ± 2.5 c 1 _+ 1 e <0.001 c 3. Mice receiving 70~g CP 4 3 ± 3 8 c 18± 16 c 7 ± 8 c 1.6 +_ 1.4 e 4. Mice receiving 7 ~xg CP 80 ± 16 65 _+ 26 35 ± 21 40 ± 12 5. Mice receiving 0.7~g CP 86± 4 62± 19 32±23 27 ± 8

Co-culture of 1 + 2 b 20± 7 e 12± 5 c 4_+ 2 ~ 0.15_+ 0.P Co-culture of 1 + 3 72 ± 14 31 ± 27 e 12 + 13 ~ 8 ± 1 e Co-culture of 1 + 4 86± 8 69± 16 32±21 34 ±11 Co-culture of 1 + 5 86± 4 69+_22 35+20 60 ± 18

a C. parvum administered IV on day 9; cells taken for restimulation on day 14 b Co-cultures initiated at cell ratio 1 : 1 c Significantly different (P < 0.05) than control d Mean ± SD of six separate experiments

Table 5. Effect of C. parvum dose on cytotoxic antibody titers

Ab response a X ± SD b

Alloimmune control 3 + 1.6 Mice given CP 700 p~g 4 + 1.0 Mice given CP 70 ~g 2.5 ± 1.7 Mice given CP 7 ~xg 1.4 + 2.2 e Mice given CP 0.7 ~xg 2.6 ± 2.1 Mice given CP 0.07 ~g 3 ± 1

a Titer defined as reciprocal of highest dilution of serum resulting in cytotoxicity greater than 10% over background. Results expressed as log z of titer b Mean _+ SD of four separate experiments cP = 0.1 as compared to control

Table 6. Effect of C.parvum given SC admixed with tumor alloantigen on primary and secondary CMC

Primary CMC CMC X _+ SD"

Spleen CMC E:T = 100:1

Day 9 Day 14

Alloimmune control 23 _+ 7 14 + 3 Admixed with C. parvum 49 _ 9 b 3 + 2 b

Secondary CMC E : T = 5 : I E : T = 1:1

A. Alloimmune control 63 + 18 30 _+ 6 B. Antigen admixed with C. parvum 13 + 2 b 3 + 1 b C0-culture of A + B 57 _+ 17 26 _+ 5

a Mean + SD of three separate experiments b Significantly different than corresponding control (P < 0.05)

spleen and mesenter ic nodes, a long with depressed in vi t ro

s econda ry responses . Suppressor cell activity, however ,

did no t deve lop (Table 6).

Discussion

The effects o f C.parvum on p r imary and seconda ry C M C

are dependen t upon dose and rou te o f adminis t ra t ion.

P r i m a r y C M C genera ted in v ivo was marked ly and com-

pa r ab ly inhibited by 700 and by 70 ~xg of C. parvum.

L o w e r doses had no effect. In contras t , inhibi t ion o f sec- o n d a r y cy to tox ic i ty (in vi tro) and genera t ion o f a cell that

suppresses seconda ry C M C in vi t ro was more pro-

n o u n c e d after 700 ~g. Similar dose dependency was

shown for C. parvum given by the IP route.

C. parvum and B C G - a c t i v a t e d m a c r o p h a g e s capable

o f suppress ing T cell funct ions have been descr ibed by

o ther inves t iga tors [7, 8, 13]. Admin i s t r a t ion o f 1,400 ~g

C. p a r v u m to no rma l mice caused the appea rance in

spleen o f phagocy t i c cells capable o f suppressing prolif-

era t ive responses to phy tohemagg lu t in in [13]. The sup-

pressor cell descr ibed by us in prev ious publ ica t ions is also

a phagocy t i c cell, in tha t its act ivi ty can be r e m o v e d by

t r ea tmen t wi th ca rbony l i ron and m a g n e t and is no t af-

fected by an t i t hymocy te serum and c o m p l e m e n t [9]. I t

differs, however , in tha t its appea rance is dependen t upon

an ongoing i m m u n e response. I f C. parvum is in jected

pr ior to a l lo immuniza t ion , suppressor cell act ivi ty is no t

detected. A p p e a r a n c e o f suppressor cells in spleen was direct ly re la ted to dose, i.e., 700 ~g caused the highest levels o f suppressor cell act ivi ty while less than 70 ~g caused

none. I t is possible tha t at h igher doses (> 700 ~g) in-

A. Lichtenstein et al.: Inhibition of Cell-mediated Immunity by Corynebacterium parvum

duction or stimulation of suppressor cells can occur independently of an ongoing immune response to alloantigen.

There are no data available on dose dependency of C. parvurn depression of other T cell functions. Scott and Warner [ 17] reported that the administration of weekly injections of low-dose C. parvum as antitumor therapy maintained normal or even increased delayed hypersensitivity reactions and responsiveness to PHA. This would be in agreement with our data showing that at lower concentrations the suppressive effects of C. parvum can be spared.

The data on route of administration indicate that splenic uptake of C. parvum is necessary for inhibition of primary and secondary CMC in spleen. Only systemic C. parvum caused this depression, while SC administration 9 days after alloimmunization was not effective.

The effects of route on PL cytotoxicity are more complex. Intravenous, but not IP, C. parvum depresses CMC measured in PLs, and the reasons for this difference are unclear. Intraperitoneal C.parvum may enhance macrophage cytotoxicity, which may override its suppressive effects on T-CMC. Intraperitoneal C.parvum is known to be very effective in causing accumulation of activated cytotoxic peritoneal macrophages [14], which would make the chromium release assay more difficult to in- terpret.

Nine days after the SC administration of C. parvum admixed with alloantigen, CMC in spleen and mesenteric nodes was increased. By day 14, however, CMC in lym- phoid organs from mice receiving alloantigen admixed with C. parvum was depressed. These results suggest a dual effect of C.parvum, i.e., immunoadjuvant in the early phase of the primary CMC response and immunosuppressive toward the later phase. Although SC C.parvum is expected to remain at its local injection site [16], enough may have become systemic to inhibit primary CMC. Alternatively, C. parvum may have leaked into the peritoneal cavity (SC injections were given in the flank) and become systemic. It took more than 9 days for suppression of CMC to occur, in contrast to the usual 5 days, and this is consistent with smaller doses gaining systemic References access. 1.

When obtained on day 14, spleen cells from these C. parvum-treated animals could not generate cytotoxicity when restimulated in vitro. However, there were no suppressor cells in the spleens of these animals. 2.

The data obtained when C. parvum was administered SC admixed with alloantigen indicate that the depression 3. of secondary cytotoxicity can occur independently of suppressor cell activity under certain conditions. Prelimi- nary data using fractions obtained after phenol-water extraction of whole C. parvum support the existence of a 4. depressive effect on secondary CMC independent of suppressor cells [ 11 ]. Phenol-extracted material is capable of

43

depressing primary and secondary CMC but does not cause the generation of suppressor cells [ 11]. Thus, while suppressor cells generated by whole C.parvum organisms may play a significant role in the depression of secondary CMC, a distinct moiety of the organism is able to depress secondary CMC without demonstrable suppressor cell generation.

Although these two phenomena are separable under certain conditions, it is not clear whether one can induce the other indirectly. In fact, under most conditions, the two effects occur together. Preliminary experiments indicate that C. parvum can prevent either priming or the in vivo clonal expansion of primed memory cells. This pre- vention of priming could cause direct depression of secondary CMC without demonstrable suppressor cell activity. At present, it is not known whether the cells responsible for this phenomenon are also involved in activation of the macrophages that suppress the generation of in vitro secondary cytotoxicity. If this is the case, it may be possible that the two phenomena are indirectly related, although separable under certain conditions.

Few studies have investigated T cell-mediated cytotoxicity in animals that have received C. parvum after the initiation of a specific immune response. Halpern [3] ex- amined in vitro cytotoxicity of lymph node cells and PLs to syngeneic tumor cells, but the effector cell in his system was unknown and could very well have been a nonspecifi- cally activated macrophage. In our original reports [9, 10], we showed that C. parvum inhibits the generation of CMC, and that phagocytic cells capable of inhibiting in vitro generation of memory CMC by control alloimmune spleen develop. The data presented here indicate that these effects are dependent upon dose and route of administration, and may be an important consideration in the design of studies in which C.parvum is to be used as an immunotherapeutic agent.

Acknowledgements: This work was supported by Grants CA-12800 and CA-09120 from the National Cancer Institute, DHEW.

Brunner KT, Engers HD, Cerottini JC (1976) The 51chromium release assay as used for the quantitative measurement of cell- mediated cytolysis in vitro. In: in vitro methods in cell-mediated and tumor immunity. Academic Press, New York, p 423 Dimitrov NV, Greenherg C, Denny T (1977) Organ distribution of Corynebaeterium parvum labeled with iodine-125. J Natl Cancer Inst 58 : 287 Halpern B, Fray A, Crepin Y, Platica O, Lorinet AM, Rabourdin A, Sparros L, Isac R (1973) Corynebacteriumparvum, a potent immunostimulant in experimental infections and in malignancies. In: Immunopotentiation. Ciba Foundation Symposium. Asso- ciated Scientific Publishers, New York, p 217 Howard JG, Biozzi G, Stiffel C, Mouton D, Liacopoulos P (1967) An analysis of the inhibitory effect of Corynebaeteriumparvum on graft-versus-host disease. Transplantation 5:1510

44 A. Lichtenstein et

5. Howard JG, Christie GH, Scott MT (1973a) Biological effects of Corynebacteriumparvum. IV. Adjuvant and inhibitory activities on B lymphocytes. Cell Immunol 7:290

6. Howard JG, Scott MT, Christie GH (1973b) Cellular mechanisms underlying the adjuvant activity of Corynebacterium parvum: Interactions of activated macrophages with T and B lymphocytes. In: Immunopotentiation. Ciba Foundation Symposium. Asso- ciated Scientific Publishers, New York, p 101

7. Klimpel GR, Henney CS (1978a) A comparison of the effects of T and macrophage-like suppressor cells on memory cell differen- tiation in vitro. J Immunol 121:749

8. Klimpel GR, Henney CS (1978b) BCG-induced suppressor cells: I. Demonstration of a macrophage-like suppressor cell that inhibits cytotoxic T cell generation in vitro. J Immunol 120:563

9. Murahata R, Zighelboim J (1979a) Inhibition of memory cell mediated cytotoxic response by systemic administration of Co- rynebacterium parvum. Cell Immunol 42:289

10. Murahata R, Zighelboim J (1979b) Inhibition of cell mediated cytotoxicity to tumor alloantigens by systemic administration of Corynebacterium parvum. Cancer Immunol Immunother (in press)

11. Murahata R, Cantrell J, Zighelboim J (1979) Differential effects of Corynebacterium parvum fractions on the immune response to

al.: Inhibition of Cell-mediated Immunity by Corynebaeterium parvum

tumor alloantigens in mice. (Abstract) In: Proceedings of the Thirteenth International Leukocyte Culture Conference

12. Scott MT (1972a) Biological effecs of the adjuvant Corynebac- terium parvum. I. Inhibition of PHA, mixed lymphocyte, and graft-versus-host reactivity. Cell Immunol 5 : 459

13. Scott MT (1972b) Biological effects of the adjuvan~ Corynebacte- rium parvum. II. Evidence for macrophage-T cell interactions. Cell Immunol 5 : 469

14. ScottMT(1974a) Corynebacteriumparvumas atherapeutic antitumor agent in mice. I. Systemic effects from intravenous injection. J Natl Cancer Inst 53:855

15. Scott MT (1974b) Depression of delayed-type hypersensitivity by Corynebacterium parvum: Mandatory role of the spleen. Cell Immunol 13 : 251

16. Scott MT, Warner SL (1976) The accumulated effects of repeated systemic or local injections of low doses of Corynebacterium parvum in mice. Cancer Res 36:1335

17. Scott MT, Milas L (1977) The distribution and persistence in vivo of Corynebacterium parvum in relation to its antitumor activity. Cancer Res 37:1673

Received August 30, 1979/Accepted November 8, 1979

inhibition of cell-mediated cytotoxicity by corynebacterium parvum

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