FURTHER INVESTIGATIONS ON THE IMMUNIZING

TRANSPLANTABLE CARCINOMA POWER OF NORMAL MOUSE TISSUES JQGAINST

SHIGEMITSU ITAMI

From the Cancer Hoepital Reeearch Institute, London, A. Leitch, Director

I. IMMUNIZING POWER OF CERTAIN FIXED TISSUES

In a preliminary communication the writer1 described a series of experiments in which various tissues were tested in respect to their immunizing power against several transplantable tumors of the mouse and rat. When these tissues were grouped according to the embryonal layers from which they arise, no correlation could be discerned between origin and immunizing power; one derivative of a layer might be an efficient immuniz- ing agent while another might be entirely inactive. There did seem to be some relation, however, between the collularity of a tissue and its immunizing power, the more cellular appearing to be more active.

For details respecting the technic employed, and for a bibliog- raphy, the reader is referred to the preliminary article.

The experiments about to be described constitute an extension of these earlier investigations. The immunizing power of muscle, for instance, has been analyzed, and it has been found that heart muscle, which is more cellular than skeletal muscle, produces a moderate degree of resistance (Fig. l), whereas skeletal muscle had been shown to be quite inactive. Myo- cardium was obtained free of blood by severing the: large vessels of the abdomen under narcosis, and then administered sub- cutaneously in the form of a hash, in doses of 0.05 cc.

A repetition of the experiment with heart muscle that had been kept for 2 days at a temperature of - 5' C. gave a similar result (Fig. 2).

As the wall of the pregnant uterus is more cellular than the 128

IMMUNIZING POWER OF NORMAL MOUSE TISSUES 129

FIO. 1. EXPERIMENT 63/302P. Moderate immunity one week after treatment with 0.05 C.C. of an emulsion of heart. Tumor dose, 0.003 gram by the needle method.

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130 SHIQEMITSU lTAMl

FIG. 2.

....

EXPERIMENT 63/306S. Moderate immunity ten days after treatment with 0.05 C.C. of an emulsion of heart kept for two daysin a refrigerator (- 5’ C.). Tumor dose, 0.003 gram by the needle method.

IMMUNIZING POWER OF NORMAL MOUSE TISSUES 131

myocardium, it might be expected that uterine muscle would prove to be at least as active in eliciting the refractory state, if the cellularity of a tissue be the only cause of its immunizing faculty. But experiment shows that the myometrium of the full-term uterus provokes no appreciable resistance even in

- 0 - c 5 - n u . * a

FIG. 3. EXPERIMENT 63/303P. Distinct immunity ten days after treatment with 0.05 C.C. of an emulsion of adult skin. Tumor dose, 0.003 gram by the needle method.

doses as large as 0.1 cc. Thus hypertrophic smooth muscle shares with striated muscle the inability to immunize against the transplanted tumor. And it is obvious, furthermore, that other factors than mere cellularity must be concerned in these differ-, ences which the various tissues exhibit in their power to call forth the resistant condition.

132 SHIGEMITSU ITAMI

FIQ. 4. EXPERIMENT 63/306S. Moderate immunity ten days after treatment with 0.05 C.C. of an emulsion of thymus kept overnight in a refrigerator (- 5' C.). Tumor dose, 0.003 gram by the needle method.

IMMUNIZING POWER OF NORMAL MOUSE TISSUES 133

The inability of the brain to immunize, which was fore- shadowed in the preliminary communication, has been amply confirmed in the present series of experiments. Fetal brain, even in amounts of 0.1 cc. has no immunizing power.

Fetal skin is one of the most efficient immunizers known, and Fig. 3 shows that the activity of adult skin is no less. This can be obtained sterile enough for practical purposes by removing the hair with 10 per cent barium sulphide, painting the bare skin with iodine, and then washing it with alcohol and ether.

It was shown in the preliminary paper that lymph-node will elicit a high degree of resistance, and it is now found that the thymus, an organ closely resembling the lymph-nodes both in structure and origin (except for its reticulum and the corpuscles of Hassall), produces an active immunity (Fig. 4).

Lung is equally efficacious (Fig. 5) , even after having been kept 1 or 2 days in the refrigerator. Thyroid, however, which is also a derivative of the entoderm, is entirely inert.

In the following table the tissues of the present series of experiments have been added to those reported in the pre- liminary paper. The sign + indicates that the tissue im- munizes, while - means that it is inert.

ECTODERY

Fetal skin. ...................................................... + Adult skin.. .................................................... + Mamma ........................................................ + Lens - Bra in - ...........................................................

.......................................................... MEBODERM

Spleen (containing blood). ........................................ + Blood .......................................................... + Testis .......................................................... + Kidney. ........................................................ + Cartilage - Bone - Skeletal muscle. - Uterinemuscle .................................................. Heart muscle. ................................................... + Lymph-node. ................................................... + Thymus ........................................................ +

....................................................... ...........................................................

................................................. -

134 SHIGEMITSU ITAMI

ENTODERM Liver ........................................................... + Lung.. ......................................................... + Thyroid. .......................................................

Embryo. ....................................................... +

- ALL LAYERS

Extraembryonic ectoderm afid mesoderm Placenta (washed free of blood). +

n 0

4

0

0

0

0

FIO. 5. EXPERIMENT 63/303P. Distinct immunity ten days after treatment with 0.05 C.C. of an emulsion of lung kept for two days in a refrigerator (- 5' C.). Tumor dose, 0.003 gram by the needle methog.

11. IMMUNIZING POWER OF THE BLOOD CELLS

There are certain definite advantages in the use of blood as an immunizing agent. It can be obtained sterile and in ade- quate amounts, it requires no preparation preliminary to injec- tion, it can be introduced into the circulation, and, finally, since

IMMUNIZING POWER OF NORMAL MOUSE TISSUES 135

the various types of corpuscle are separable one from the other with comparative ease, information may be gained in respect to the immunizing power of the different groups.

The question of blood grouping in relation to immunity may be neglected, as it has been shown that neither rats nor mice show isoagglutinins and isohemolysins (Rohdenburg,2 Mac- Dowel1 and Hubbard,s Epstein 4).

A survey of the literature shows that the amount of blood required to immunize a mouse (0.3 to 0.5 cc.) is large as com- pared with the dose of fixed tissues (0.05 cc.), and that the degree of resistance which it elicits is not high.

So far as the writer is aware, no analysis of the immunizing power of the various types of blood-cell has yet been published, with the exception of a paper by Sittenfield,6 who demonstrated that a leucocyte cream containing an increased proportion of lymphocytes (i.e., about 25 per cent) does not immunize against tumor transplan tation.

The blood employed in the present experiments was mixed immediately after withdrawal with an equal volume of 1 per cent sodium citrate in physiological saline solution. Of this suspension 0.1 cc. was injected into the tail-vein, representing 0.05 cc. of blood. Mice will easily tolerate such an amount, and, indeed, even as much as 2 cc. of physiological saline solution can be introduced into the circulation at one time without apparent bad effect. Figs. 6 and 7 show that 0.05 cc. of blood causes some resistance.

It is an interesting fact that washed red corpuscles even up to the amount obtainable from 0.2 cc. of blood have no immuniz- ing power. The experiment has been repeated several times, and with different washing fluids, but always with the same negative outcome pictured in Fig. 8, which illustrates a typical experiment of this sort. Thus the resistance elicited, if any, by 0.05 cc. of blood must depend upon some element other than the erythrocyte.

It was hoped that some light could be thrown on this question by producing nucleated red cells in the blood of the mouse, according to the method described by Eddy and Brown;

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FIQ. 6. EXPERIMENT 63/395S. Slight immunity four days after intravasoular treatment with 0.05 C.C. of whole blood. Tumor dose 0.003 gre,m by the needle method.

IMMUNIZING POWER OF NORMAL MOUSE TISSUES 137

lMMUNlZED CONTROK

b * . . ' ' ' ' . - . I

I @ CM FIQ. 7. EXPERIMENT 63/297R. Moderate immunity eight days after intra-

vascular treatment with 0.05 C.C. of whole blood. Tumor dose, 0.003 gram by the needle method.

Ro. 8. EXPERIMENT 63/299P. No immunity eight days after intravascular treatment with washed erythrocytes obtained from 0.2 C.C. of blood. Tumor dose, 0.003 gram by the needle method.

IMMUNIZING POWER OF NOIWAL MOUBE TISSUES 139

but although their technic was followed exactly in every par- ticular the attempt to obtain young erythrocytes failed.

When one reflects on the observation just cited, which seems to show that citrated whole blood will immunize, whereas washed red cells will not, it seems impossible that the small

FIQ. 9. EXPERIMENT 63/298Q. Moderate immunity five days after intravascular treatment with washed leucocytes obtained from 1 C.C. of blood. Tumor dose, 0.003 gram by the needle method.

number of leucocytes contained in 0.05 cc. of blood should suffice to elicit resistance, even when introduced directly into the circulation. That the leucocyte is effective in sufficient amounts, however, is shown by Fig. 9, where mice were injected with the amount of leucocytes obtainable from 1 cc. of blood.

140 SHIOEMITSU ITAMI

These cells were isolated after the method of Hamburger and Hekma 'I from citrated blood, and finally suspended in an appro- priate amount of saline.

L

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.I

Ir(

L

- FIQ. 10. EXPERIMENT 63/300R. Distinct immunity five days after intravascular

treatment with thymic cells obtained from 0.05 gram of thymus. Tumor dose, 0.003 gram by the needle method.

The lymphocytes were obtained from the thymus and lymph- nodes except in two experiments (Figs. 11 and 14); those of the spleen cannot be separated from the other cells of this organ. The tissue was minced and the lymphocytes which had thus been freed were suspended in fresh rat serum, in order that the effects of any tissue coagulin present might be neutralized. A11 the lymphocytes in lymph-nodes or thymus cannot be liberated

IMMUNIZING POWER OF NORMAL MOUSE TISSUES 141

in this way, and it was estimated that the yield represented but about one eighth of the amount of tissue employed; that is to say, 0.05 gm. of. tissue would furnish approximately 0.006 gm. of isolated lymphocytes.

7

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FIG. 11. EXPERIMENT 63/304T. Distinct immunity five days after intravascular treatment with splenic cells obtained from 0.05 gram of spleen. Tumor dose, 0.003 gram by needle method.

Rat serum was used because it is obtainable in larger amounts than mouse serum. It is well known that alien tissues have no immunizing power, so that any resistance elicited by the lymphocyte suspension can be definitely ascribed to the lympho- cytes therein. That this cell can immunize when introduced into the circulation in doses of 0.006 gm. is shown in Figs. 10, 11, 12, 13, and 14.

FIG. 12. EXP~CRIMENT 63/299P. Moderate immunity five days after intra- vascular treatment with lymphatic cells obtained from 0.06 gram of lymph node. Tumor dose, 0.003 gram by needle method.

IMMUNIZING POWER OF NORMAL MOUSE TISSUES 143

FIO. 13. EXPERIMENT 63/295U. Slight immunity three days after intravascular treatment with lymphatio cells obtained from 0.05 gram of lymph node. Tumor dose, 0.003 gram by the needle method.

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FIQ. 14. EXPERIMENT 63/296Q. Slight immunity three days after intravasoular treatment with splenio cells obtained from 0.05 gram of spleen. 'Tumor dose, 0.003 gram by the needle method.

IMMUNIZING POWER OF NORMAL MOUSE TISSUES 145

When the immunizing power of the dose of lymphocytes obtainable from 0.25 gm. of thymus or lymph-node was com- pared with that induced by the quantity of white cells that could be collected from 5 cc. of blood, the same amount of serum having been used for suspension in each case, it appeared that the lymphocyte is more effective than the other leucocyte.

SUMMARY

1. Resistance to tumor transplantation can be brought about by previous treatment with some, but not all, of the derivatives of all three germ layers.

2. Analysis of the immunizing power of the blood cells shows that washed erythrocytes are ineffective, and that both the granular leucocytes and lymphocytes will elicit resistance, the latter being somewhat more efficient than the former.

REFERENCES 1. ITAMI: J. Cancer Res., 1919, iv, 23. 2. ROEDENBURG: Proc. SOC. Exp. Biol. and Med., 1920, xvii, 82. 3. MACDOWELL AND HUBBARD: Proc. SOC. Exp. Biol. and Med., 1922, xx, 93. 4. EPSTEIN: Progres mBd., 1924, No. 19, 296. 5. SITTENFIELD: J. Cancer Res., 1917, ii, 151. 6. EDDY AND BROWN: Amer. Jour. Physiol., 1922, Ixii, 242. 7. HAMBURGER AND HEKMA: Biochem. Ztschrft., 1907, iii, 88.

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