Download iiiiiiiiii - Cancer Research

AGGLUTINATION TESTS IN THE STUDY OF TUMOUR
IMMUNITY, NATURAL AND ACQUIRED
THOMAS LUMSDEN, M.D.
Director of Cancer Research, London Hospital
In 1898 Bordet (1) recorded the fact that an animal immunised against
red blood cells from a different species developed in its serum lysins and
agglutinins more or less specific to the red cells used as an antigen. Two
years later Ehrlich and Morgenroth (2), working with goats, showed that even
the inoculation of homologous red cells in some cases evoked haernolysins
(Isolysins ). Todd and White carried these investigations further and in
TAIlLE
I: Correlating the Various Antibodies Toxic to Cells and Showing Their Nomenclature
Cytotoxins
I
I
I
I
Surface cytoplasmic antibodies
,_I,
Ixo-
I
I
I
Cyto- or
Chromato,
Iysins
Agglutinins
H aemolysins
I Ietero-
Nuclear
antibodies
I
I
I
Hetero-
Iso-
Species
I
I_I,
Iso-
------
I
I
for Nucleated
cells
Cell agglutinins
for Blood
celts
I Inemagglut i nl ns
I
Hetero-
Auto(autospermatoxi ns)
Iso-agalutinins
~I
Hetero-
I
Antispecies
bodies
\
Non-species
specific
I
lIeterocytotoxic
(heterotoxins)
Homo-
I
Hornocytotoxic
(homotoxins)
When nucleated homologous cells are used as an antigen, the surface and the nuclear antibodies (agglutinins and chromatolysins) run roughly parallel.
1910 (3) discovered that in oxen isolysins varied in their haemolytic effect on
the red cells of different individuals; in short no two isolytic sera were absolutely alike, their characters depending upon two factors: (a) the individuality
of the injected corpuscles; (b) the individuality of the animal into which they
are injected. By absorption of polyvalent sera, Todd and White were able to
identify the cells of any individual ox. Later (1930) Todd (4) in an important paper confirmed in fowls the results obtained in oxen, having found that
by means of simple immunity reactions he could differentiate the red corpuscles of any particular fowls which were not close blood relations.
The experiments described below show that in normal rats the agglutinability of the red blood corpuscles varies widely, and that a high degree of
agglutinin sensitivity is characteristic of certain individual rats not necessarily
of any particular breed or group.
395
396
THOMAS LUMSDEN
The reactions of a number of sera of rats immunised against various normal and malignant tissues with readily agglutinable rat red blood cells have
also been investigated, and are considered in the light of results previously
recorded (5, 6, 7). The various antibodies toxic to cells are tabulated, and
the nomenclature used in this paper is given, in Table 1.
METHODS
Sera are obtained from a few drops of blood drawn into fine glass tubules
from the tails of the rats under investigation. One end of each tubule is
sealed and after centrifugation the portion of the tubule containing the serum
is cut off with a diamond.
In order to obtain a suitable suspension of red blood cells, a counted number of drops of blood from a glass tubule (1. 5 mm. in diameter) are run directly into a glass centrifuge tube containing about three times as much
citrated saline. The cells are washed three times with saline and centrifuged
practically dry; as many drops of saline as there were originally of blood are
added and, after agitation, a sample is examined in the serum of the rat from
which the cells came in order to ascertain that it is entirely free from cell
clumps. There is a good deal of variation in the ease with which a completely homogeneous dispersion of cells can be obtained, and in some instances
it may be found best to avoid the saline washings and the repeated centrifugation thus entailed. Indeed; saline washing has been found to be not really
essential, although it was done in the experiments recorded.
The drop of the serum under investigation is run from the tubule on to a
grease-free cover slip, an appropriate amount (about a quarter of a drop) of
citrated red blood cells is added from a very fine capillary tube and is intimately intermixed with the serum. The cover slip is then inverted over a
hollow slide and sealed with paraffin. It will be seen that there is little dilution of the serum and all the tests are therefore roughly comparable.
The hanging drop preparations are placed in a thermostat at 37 0 for
twenty minutes or are left at laboratory temperature for two to three hours.
Thereafter the agglutination, if any, is estimated, first with the naked eye, the
slide being tilted gently so that the corpuscles flow from side to side. In the
presence of macroscopic agglutination the corpuscles do not flow freely, but
tend to form a mass sharply demarcated from the serum. The hand holding
the slide (now turned face downwards) is then made to describe a circle, ten
or twelve times, as rapidly as may be without swinging off the drop, and the
titre of agglutination is estimated microscopically, with a % inch objective.
The results are tabulated in accordance with the following scale. In recording
these, wherever doubt existed as to the degree of agglutination the lower estimate was assigned.
(1)
(2)
(3)
(4)
(5)
+++
++ ±
++
+ ++ ..
.
Complete agglutination; no free red corpuscles seen, all being
aggregated in very large masses.
.No free red blood corpuscles; clumps smaller but still large.
... Some free red blood corpuscles now seen, between large clumps.
.. A halfway stage between (3) and (5).
. .Agglutinated masses just visible to the naked eye.
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
(6) ±
(7) ;t
(8) :;l;
(9) 0
397
.. The coarsest agglutination not visible to the naked eye.
... Clumps small though definite, consisting of 10 to SO corpuscles
or thereabouts.
.Unusually marked rouleau formation with few if any true clumps.
Complete absence of agglutinative effect when compared with the
same suspension of cells mixed with autogenous serum.
The sera should be free from haemoglobin as its presence may cause slight
clumping in the absence of agglutinins. "Milky" sera are also useless, since
they sometimes give rise to haemolysis masking any agglutination which may
have taken place. The use of the author's tissue culture table (8) greatly
facilitates the performance of these tests.
All the highly immunised rat sera were produced by weekly or fortnightly
intraperitoneal injections of 1 c.c. of the antigen named and these inoculations
were repeated at least five, and in some cases fifteen to twenty, times.
In order to repeat or to extend these experiments, it is necessary to find a rat or rats
with readily agglutinable corpuscles (see below). To do this, immunise several tumourresistant (non-take) rats by repeated weekly intraperitoneal inoculations of 1 C.c. of Jensen
rat sarcoma (J. R. S.) fragments for two months. Draw some blood from the tails of these
rats; the sera are certain to contain agglutinins, as are the sera of rats highly immunised
against rat spleen. Prepare washed red blood corpuscles, using the technique already described. Test the sera of these immunised rats against the washed red cells from 20 to 30
normal male rats of various breeds (other than pure albino Wistar rats). Table V shows
that it should not be difficult to find a few suitable rats. Once found, such rats can be used
indefinitely for the supply of the necessary agglutinin-sensitive corpuscles.
Since the agglutinability of the red blood corpuscles of different agglutininsensitive rats may vary slightly in degree in the response to particular immune sera (Table XVI), it is best in any series of experiments to use the same
individual rat as the source of the corpuscles to be used as a test reagent. In
the following experiments the corpuscles were always taken from an agglutininsensitive cream hooded rat, marked R. S., except where otherwise stated.
As mentioned below, agglutinins always appear in the sera of rats in which
a tumour implant regresses, but in some cases the agglutinins never reach a
high titre and may be demonstrable only for a few days. It is therefore essential that daily examinations be made in such experiments.
EXPERIMENTAL OBSERVATIONS
(1) The question under investigation when the observation (Table IV)
which proved to be the source of this paper was made, was whether the sera
of rats immunised against normal or malignant homologous cells were isohaemolytic.
Table II shows the results in one of many experiments which invariably
agreed in indicating that isohaemolysins are not present in the sera of rats
immunised against Jensen rat sarcoma, rat spleen, rat testis, or rat blood
corpuscles. In fact, so far as the evidence goes it seems that rats are incapable of forming isohaemolysins.
Table III demonstrates that there is no haemolysis in vitro, even when red
corpuscles highly sensitive to iso-agglutinins (see below) are concerned.
TABLE
II: One of Many Experiments Showing Absence of Iso-haemolysins in Rats
Serum of Rat Immune to ].R.S.
Red Blood Cel1s
of Normal Rat
Diluted 1 in 20
with Saline
Guinea-pig
Serum Diluted
1 in.5 with
Saline
R.C. 17: 1 part
17: 1 part
B.C. 17: 1 part
Saline control: 1 part
Normal serum control: 1 part
1 part
1 part
1 part
1 part
1 part
1 part
1 part
1 part
1 part
1 part
t.c.
TABLE
Haernolysis
o
o
o
()
o
Ill: Showing that Sera of Rats Immunised against Various Homologous Tissues Agglutinate
but Do Not Lyse the Red Blood Corpuscles of Agglutinin-sensitive Rats
Washed Red
Corpuscles of
AgRlutininsensitive
Rat, R.S.
(I in 3 of Saline)
1. RR.Cs.
1 part
2. R.B.Cs.
1 part
3. R.B.Cs.
1 part
4. R.B.Cs.
1 part
5. R.B.Cs.
1 part
6. R.B.Cs.
I part
7. R.B.Cs.
1 part
8. R.B.Cs.
1 part
9. R.B.Cs.
1 part
TABLE
Rat Serum
Complement
].R.S.-immune rat, R.C.
2 parts
J,R.S.-immune rat, R.C.
2 parts
].R.S.-immune rat, LC.
2 parts
J, R.S.-immune rat, B.C.
2 parts
J,R.S.-immune rat, R.S.
2 parts
Rat-blood-immune rat,
R.C. 2 parts
Rat-blood-immune rat,
r..c, 2 parts
Normal rat
2 parts
None
None
Guinea-pig serum
1 part
Guinea-pig serum
1 part
Guinea-pig serum
I part
Guinea-pig serum
1 part
Guinea-pig serum
1 part
Guinea-pig serum
1 part
Guinea-pig serum
I part
Guinea-pig serum
1 part
Agglutination
Haemolysis
+++
+++
+++
++
+++
++
+
o
0
o
0
0
0
0
±
0
+
0
._-
IV: Recording the Experiment in Which It Was Revealed. that the Red Blood Corpuscles of
a Certain Indioidua! Rat Had an Agglutilzin Sensitivity Far Above the Average
Breed of Rat from
Which Washed
Cells Were Taken
1. Fawn
2. Black
3.
4.
5.
6.
Agouti
Cream
Stone
Stone Hooded
7. Cream Hooded
8. Agouti Hooded
9. Fawn Hooded
Serum of ].R.S.-immune
Rat, R.C., 19th
Serum of ].R.S.-immune
Rat, R.C.L.S., 18th
Lysis
AgRlutination
Lysis
Agghitination
o
o
o
o
o
o
o
o
o
o
o
o
o
+++
o
o
o
o
o
o
o
398
o
o
o
o
o
o
o
()
o
o
o
()
+++
()
o
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
TABLE
Breed
Cream
Hooded
Males
1 R.S.
2
3
4
5
6 R.C.
7 L.C.
Cream
Hooded
Females
I
2
3
4
5
6
7
8
9
10
11
12
Fawn
Males
I
2 L.S.
3 U.
4
5 L.C.
6R.C.
7
Fawn
Females
I
2
3
4 B.S.
5
6 R.S.
7
8
9
10
11
12
399
V: Assay of the Agglutinability of the Red Blood Corpuscles of 187 Rats of 12 Breeds."
Result
+++
0
0
0
0
+
+
±
0
0
0
0
0
0
0
0
0
0
0
0
+++
±
0
+
+++
0
0
0
0
+
0
+
0
0
0
0
0
0
Breed
Black
Males
1
2
3 L.S.
4
5 L.C.
6 R.C.
7
Black
Females
t R.S.
2 L.S.
3 B.S.
4
5
6
7
8
9
10
11
12
Stone
Hooded
Males
IU.
2 B.S.
3L.S.
Stone
Hooded
Females
I
2
3
4
Fawn
Hooded
Males
I
2
3
4
5
6
7
Result
0
0
+
0
+
++
0
++
+
±
0
0
0
0
0
0
0
0
0
0
+++
0
0
0
0
0
Breed
Agouti
Hooded
Males
1
2
3
4
5 R.C.
6 L.C.
7
+
0
0
0
0
0
+
+++
0
Stone
Males
1
2
3
0
0
0
Cream
Males
1
2
3
0
0
0
Agouti
Males
I L.S.
2
3
LISTER
Males and
Females
I
2
3
4
5
6
7
8
9
10
11
0
0
0
0
0
Result
12
13
14
15
16
17
18
19
+
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
0
0
0
Breed
LISTER
(cont.)
20
21
22
23
24,
25
26
27
28
29
30
31
32
33
34
35
36
LONDON
LISTER
Males and
Females
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Result
Breed
Result
LONDON
LISTER
±
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
±
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
+
(cont.)
28
29
30
31
32
33
34
35
36
WISTAR
Males and
Females
1
2
3
4
5
6
7
S
9
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
0
0
±
0
0
0
0
0
±
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
• In this table titres lower than ± were disregarded since the only object of the experiment
was to find rats in which the red cells were exceptionally sensitive to agglutinins.
(2) In order to determine whether there were different blood groups in
rats which might be important in relation to the existence of isohaemolysins,
the sera and corpuscles of 28 normal rats of twelve different breeds were
tested in batches of 6, 6, 6, 6, and 4, and each against each (180 separate
tests). In not one instance did the serum of a normal rat cause agglutination
of the red blood corpuscles of any other normal rat. It seems, therefore, that
there are no characteristic blood groups in rats.
(3) Although normal rat sera never agglutinate rat red cells, it was
thought worth while to see whether the sera of rats immunised against Jensen
sarcoma would do so. This investigation produced some unexpected results.
In the first experiment the sera of two rats immune to Jensen sarcoma were
each separately mixed with the washed corpuscles of nine normal rats, each
of a different breed. Table IV shows that, while lysis did not occur in any of
the tests, agglutination was complete in the case of the corpuscles of rat no. 7,
i.e. male cream hooded rat (R. S.), while it was entirely absent in the 8 other
rats, with both the immune sera tested.
400
THOMAS LUMSDEN
TABLE VI: Showing tha; Over a Period of Four Months the Degree of Agglutinin Sensitivity of the
Red Blood Corpuscles of the Individual RaJs Named Was Practically Constant
Serum of R.C.
19th, Applied
Aug. 25, '37:
Agglutination
Resulting
Red Blood
Corpuscles of
MALES
R.S. Cream Hooded
R.C. Fawn
L.S. Fawn
R.C. Black
L.S. Black
R.C. Agouti Hooded
L.C. Agouti Hooded
B.S. Stone Hooded
FEMALES
L.S. Black
R.S. Black
Same Serum;
Same R.B.C.:
Results
Oct. 29, '37
Same Serum;
Same R.B.C.:
Results
Dec. 12, '37
+++
++±
+++
++
+
+
+++
+++
±
+
+++
+++
++±
++
+±
dead
+++
+++
+++
+++
+++
++
+
+
+++
+++
+
++
+
+
TABLE VII: Showing Inheruance of Agglutinin Sensitivity
Titre of Agglutinogens in the Red Blood Corpuscles of Offspring (Born Nov. 1, '37)
of R.S. Cream Hooded Agglutinin-sensitive Male Rat
Date
(1937)
Nov. 14
Nov. 26
Dec. 7
Dec. 28
R.e.
Male
++
++
++±
++±
B.C.
Male
0
++±
++±
++±
L.S.
Male
++
++
++±
++±
B.S.
L.C.
R.C.L.S. L.e.R.S. R.S.
Male
Female Female Female Female
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Offspring (Born
Nov. 27, '37) of
Stone Hooded
Agglutininsensitive
Male Rat
Offspring (Born Nov. 7, '37) of Fawn Male
Agglutinin-sensitive Male Rat
Date
Nov. 14
Nov. 26
Dec. 12
Dec. 28
R.e.
Male
L.e.
Male
B.C.
Male
++±
++±
++±
++±
+±
++
++±
++±
++±
++±
++±
++±
L.S.
Male
0
0
0
0
R.S.
R.C. L.e.
Female Male
0
0
0
0
++
0
0
++
B.e.
±
++
(4) In order to determine whether this isoagglutinability was characteristic of this particular breed of rat, the corpuscles of six other male and twelve
female cream hooded rats were tested, giving the results shown in Table V.
It will be seen that in two of the males (R. C. and L. C.) other than R. S. the
corpuscles were agglutinated definitely though not completely, while only one
of the twelve specimens from females showed feeble agglutination. It is clear
that in this breed agglutinability is an individual and not an invariable racial
characteristic; it appears to be more usual in males than in females.
To ascertain the frequency of agglutinin sensitivity in the red blood corpus-
401
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
TABLE VIII: Recording a Number of Indiflidual Experiments and Showing That in Rats Immunised
against Homologous Nudeated Tissues the TitHS of Chromatolysins and of Agglutinins
Run Roughly Parallel. This is Not So When Non-nucleated Cells
(Red Blood Corpuscles) Are the Antigen
Rats
Degree of
Degree of
Percentage Agglutination Agglutiof ].R.S. of Agglutinin- nation of
Cells
sensitive
Ordinary
Rat
Unselected
Killed
R.B.Cs. Rat R.B.Cs.
Rats
1
2
3
4
5
6
7
8
Anti-Rat-Testis Rats
30
+++
20
++±
15
+++
15
++±
10
+
2
+
±
2
0
±
0
0
0
0
0
0
0
0
1
2
3
4
5
6
7
8
9
10
Anti-Rat-Blood Rats
5
+++
2
+±
2
0
1
+++
0
1
0
+++
0
+++
0
+±
0
±
0
±
0
0
0
0
0
0
0
0
0
0
l.R.S. Immune Rats
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
80
70
70
35
30
30
30
25
20
20
20
20
15
15
10
10
5
5
5
2
1
0
+++
+++
+++
+++
+++
+++
++±
++±
++±
++±
++±
++±
++±
++
+±
+±
+±
+±
+
+±
±
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Degree of
Degree of
Percentage Agglutination Agglutiof ].R.S. of Agglutinin- nation of
Cells
sensitive
Ordinary
Rat
Unselected
Killed
R.B.Cs. Rat R.B.Cs.
cles of rats, over 200 individuals of various breeds were examined in addition
to those mentioned above. Most of the results are shown in Table VI but
since this table was made during a search for rats with very highly agglutininsensitive red blood corpuscles, degrees of agglutination under ± are not recorded. Agglutinability of a grade below this is relatively common, and is by
no means unimportant (Tables XX-XXII). While the degree of sensitivity
to iso-agglutinins certainly varies from individual rat to rat, agglutinability
will be seen to be commoner in some breeds than in others. Not one of the
36 Wistar rats tested showed this reaction in marked degree and it is comparatively rare in Lister Institute and London Hospital black and white rats.
The numbers tested do not allow any reliable comparison to be made in the
male rats of the other breeds which were tested.
Iso-agglutinin sensitivity continues unaltered in degree certainly for four
months (see Table VI) and possibly during life. This constancy is fortunate,
since once a rat with agglutinin-sensitive red cells is found its corpuscles can
be used as a test reagent for the presence and titre of agglutinins in any number of experiments.
(5) Iso-agglutinin sensitivity appears to. be markedly hereditary for, as
may be seen from Table VII, of sixteen young rats bred from three agglutinin-
402
TABLE
THOMAS LUMSDEN
IX; Showing That, When the Highest Titre of Iso-agglutinins and of Chromatolysins Observed
in the Course of Many Examinations of the Sera of the Immune Rats Named Are
Compared, They Run Roughly Parallel Except When Non-nucleated
Cells (Blood Corpuscles) Are Used as the Antigen
Sera of
Rats
Immune
to ].RS.
Highest
Titre of
Chromatolysins
Highest
Titre of
Agglutinins
(+++ =100%)
Sera of
Rats
Immune to
Rat Spleen
Highest
Titre of
Chrornatolysins
Highest
Titre
of
Agglutinins
RC. 19
I..C. 18
L.C. 19
RS. 19
I..S.R.C.18
B.C. 18
R.C.18
B.C. 19
90
90
90
70
65
60
50
40
+++
+++
+++
+++
+++
+++
+++
+++
I..S.
B.C.
R.S.
I..C.
R.C.
80
70
35
10
15
+++
+++
+++
++±
++
Average
69
3
Average
42
2.7
Sera of
Rats
Immune to
Rat Testis
Highest
Titre of
Chromatolysing
Highest
Titre
of
Agglutinins
Sera of
Rats
Immune to
Rat Blood
Highest
Titre of
Chromatolysins
Highest
Titre
of
Agglutinins
L.e.e.
RC.C.
RC.I..
L.e.I..
60
50
10
5
+++
++±
+
±
I..e.C.H.
I..C.L.
R.e.B.
+++
+±
R.C.L.
L.e.B.
5
5
2
2
2
1
Average
2.8
Average
31
1.75
n.c.c.a.
0
±
+++
±
Sera of Rats
Immune to
Rat Serum
Highest
Titre of
Chromatol ysins
Highest
Titre of
Agglutinins
n.c.c.n.
o
o
o
o
o
o
o
o
o
o
o
o
L.C.C.H.
RC.F.
L.C.F.
RC.B.
L.e.B.
1.4
sensitive males and females of corresponding breed in which agglutinability
was less marked, eight have highly agglutinable red blood corpuscles.
(6) Tables VIII and IX show that the red corpuscles of the agglutininsensitive rat (R. S. cream hooded) were agglutinated not only by the particular
immune sera originally tested (see Table IV), but by the serum of every rat
which had been immunised against homologous nucleated cells, whether normal (spleen, testis) or malignant (Jensen rat sarcoma).
(7) Table VIII shows also that though these immune rat sera agglutinate
the corpuscles of agglutinin-sensitive rats, they are unable to cause agglutination of the red cells of other normal rats.
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
403
(8) Table IX records the highest titre of both chromatolysins and agglutinins ever found after many tests in the particular immunised rats named.
Here, as in Table VIII, it is seen that the titre of agglutinins runs closely
parallel with the titre of chromatolysins, but only when the antigenic cells are
nucleated. Rats immunised against rat red cells develop agglutinins, but
little if any chromatolysins.
(9) Comparison of the titre of agglutinins in the sera recorded in Tables
VIII and IX shows that Jensen rat sarcoma, rat spleen, rat testis and rat blood
may be placed in this order of efficiency as agglutinogens and in the same
order as agents in the production of homocytotoxins (chromatolysins ) (8).
Red cells are by no means invariably effective even as haemagglutinogens. It
appears that the more invasive the type of cell, the higher its activity as an
antigen. It must be noted that the correspondence between the titres of isoTABLE
X: Showing That Iso-agglutinins Always Appear in the Sera of Rats in Which a Tumour
Implant Regresses, but That Agglutinins Are Never Demonstrable in the Serum of
a Rat in Which a Tumour is Growing Progressively
(See also Tables XI-XIII)
Day55
1
2
;)
4
5
6
7
8
9
-
--
7
0
0
0
0
0
0
0
0
0
0
0
--_.- ..._
0 ... 0
..
0
0
0
0
- ------ --- -- -~+
0
0
-- - - - -. --
10
11
12 -
0
+
0
0
0
-
1~
11
9
0
+
0
+
... ..
•••
.-
0 ••
0
++
-
+
-
-
1++
-
t+
0
-
++
+
-
+t
-
++
+
--
++
U-L.1..J
++t
++
+-+
++
+
--
-
to
---
em. 5Ca1.e
0
0
~+~
+
++~
t,.~
+
+tt
+
-+Ht
+
agglutinins and homotoxins may be somewhat obscured in sera with high titres
because, when the chromatolysins reach a titre of 30 or 40 per cent, the agglutinins accompanying them are already capable of causing complete agglutination, so that above this point the rise in titre is not shown and the sera
must be titrated by dilution in order to demonstrate the parallelism of the
titres of the two antibodies. Further, when the homocytotoxins are undetectable-for example, long after the last injection of antigen-agglutinins
may still persist in effective degree. Within reasonable limits, however, there
seems no doubt that the titre of agglutinins may be taken as a rough index of
the existence and amount of chromatolytic homocytotoxins, The probable
reason for this parallelism is discussed later.
(10) We now come to investigate whether the chromatolysins and agglutinins shown above to exist in the sera of rats immunised against homologous nucleated cells have any relation to the mechanism by which rats Ire-
404
THOMAS LUMSDEN
quently resist the invasion of implanted tumour cells, as for instance when
" spontaneous regression" takes place. To this end the sera of over 200 rats
into which 0.1 c.c, of Jensen sarcoma fragments had been implanted were examined every two days or so from five to seven days after the implantation
until the fate of the implant was certain. The results of some typical experiments are recorded in Tables X-XIlI.
The most significant conclusions which may be drawn from these experiments are as follows:
(a) When a tumour grows progressively, agglutinins are absent or undetectable by the methods used.
TABLE
XI: Agglutinins in Sera of Animals Implanted with Jensen Rat Sarcoma
(See Table X)
-..... -... ....-- -- ---- --- ---- -- ----- --..
--
~aY5 5
1
2
3
4
5
6
7
8
9
10
11
12
7
9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
0
0
0
-
0
0
0
0
0
-
..- ••
11
0
+
0
++
0
0
0
++
0
0
13
0
---
--
+-
++
++
+-+
+
---
--
--
0
ttt
Ht
++
Hot
:I:
++
+
H·
+
+ +
+++
++
++ ~ +
+ It-m. 5Cale ±
---
(b) In every case when a tumour fails to grow or regresses spontaneously,
at some time or other agglutinins can be demonstrated in the serum of the rat
concerned. It must be insisted that frequent examinations (daily or every
two days) are essential because the agglutinins may appear only for a brief
period. Their titre rises, so as to be demonstrable, during or after regression, but it varies much in height in different individual rats and varies also in
the length of time during which it persists, so that the occurrence of these
antibodies may be overlooked if only a few examinations are made, say at
weekly intervals.
These findings confirm the results of cytotoxin experiments in which tissue
cultures were the test material,' but the agglutination tests have the great advantage that they are easy to perform and to repeat, and the results are unmistakable and can be estimated without special training. The tissue culture
experiments, on the other hand, require for their performance a considerable
degree of skill and experience.
While carrying out the experiments recorded above it became possible,
after a time, to judge with absolute confidence from serological examination
what the fate of any particular tumour implantation had been, without seeing
1 For an example of such an experiment recently published by Lumsden and Phelps (6), see
Table XIV.
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
405
the rat from which the serum under investigation had been taken, and the
conviction that there was a radical relation between the presence or absence
of these antibodies and the fate of the tumour was forced upon the observer.
(11) It had already been found in the search for blood groups that the
corpuscles of the agglutinin-sensitive male rat R. S. cream hooded were not
agglutinated by normal rat serum, but to make quite certain of this point the
sera of 75 normal rats from twelve different breeds were mixed, as above, with
the red blood corpuscles of the R. S. cream hooded male and in not one instance did agglutination take place. It is clear, therefore, that the sera of
normal rats do not contain iso-agglutinins.
TABLE
XII: Agglutinins in Sera of Animals Implanted with Jensen Rat Sarcoma
(See Table X)
.. C. .. e.... ...-- ..
..
-..
--.. .... 0" ..
.. ...
..
.....
...
pays 7
1
2
3
4
.5
6
7
8
9
10
11
12
9
11
0
0
0
0
O
0
." 0
0
4II1II' 0
0
0
0
0
0
0
0
0
." 0
0
0
0
0
0
0
0
0
0
..- ---- --
15
13
0
0
0
0
0
0
0
0
0
-
+
aD
-
-
0
++
± -- +±
± ±
±
--
±
-
±
±
----
+t
+
--
±
±
---
0
..
-----
±
+.±
+
+
+
l-L....I.-J-J
± cm.ecete +
As might be expected when the sera of rats whose red blood corpuscles are
agglutinin-sensitive are mixed with corpuscles of other similar rats, no agglutination takes place, and similarly the sera of rats immune to Jensen sarcoma do not agglutinate the red blood cells of other rats immune to that tumour. In other words, iso-agglutinins are not present in the sera of rats whose
red blood corpuscles contain much agglutinogen, and conversely, in rats having a high titre of iso-agglutinins in their sera, agglutinogens are absent from
the red blood corpuscles (Tables XVII, XX-XXIII).
(12) Since the sera of rats immunised against homologous tissues (i.e.
containing homotoxins) invariably agglutinate the corpuscles of agglutininsensitive rats it was of interest to determine whether the sera of rats immunised
against heterologous tissues containing heterotoxins and anti-species bodies,
but not homocytotoxins, would agglutinate these corpuscles. Here, as with
TABLE
XIII: Agglutinins in Sera of Animals Implanted with Jensen Rat Sarcoma
(See Table X)
..
... .. - ...
...
9
Dcrye 1
....
0
0
0
0
0
0
0
0
~
3 ... 0
0
~ 0
0
0
0
0
1
2
....- ........
-
4
5
•
--
-...- ..-
6
7
0
0
8
0
..... t+0 --11 -12 -- + -9
10
~
TABLE
1~
11
~
..... ..
a
0
0
0
0
F
•
0
0
------
±
++
0
++
0
1:
'tt
+
tt
0
+
--
+
0
•
0
--
ott
----em eceie
't't
0
ttt
0
++
~
XIV: Showing the Fate of Tumour Grafts in Ten Rats and the Results 01 Tissue Culture
Tests of the Rats' Sera *
7
8
<17· 0
0
Day 6
26
-- -...
2~ <1730
31
32
33
34
0
0
.....
- .3
10
12
13
<liT.
0
0
-0
0
0
........
.. ...
...
----...... ....
.., ... 0
~
0
~
0
0
0
0
0
0
....- -..
.. -- .....
0
0
0
,.
lifo
0
0
0
0
0
...-
0
0
0
t
0
1~
570
~c ~
1%
- .... ........ £
--
.-~
~
157-
.....1'70 . .~70.
-". 27-.'. ,. .. .
0
0
0
35 -'"
38 <1% <1%
0
37 0
* The percentage
~
9
.
-
1070
~7a
..57-
!'70
)~7.
~io
"
,
Cm.O
---
~07a
I
I
5
10
4570
-:;d
1570~
•
"3
m
<!'70 170
• tJI
<lit. 470 o'
• :3
.
0
0
til
·
above the silhouette represents in each instance the proportion of emigrated
cells killed when the serum of the rat concerned was applied to a one-day culture of J. R. s.
Chromatolysins always appear at some stage when regression takes place j they are never present
or are negligible in amount when a tumour grows progressively.
406
407
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
TABLE
XV: Showing the Titre of Iso-agglutinins and of Chromatolysins in the Sera of Rats Immunised
against Various Homologous Nucleated Cells and in the Euglobulin Fraction of the
Same Redissolved in an Equivalent Volume of Normal Rat Serum
Agglutination
Chromatol ysis
Immune Rat Sera
Effect of
Serum
(+++=100%)
Effect of
Euglobulin
Fraction
Effect of
Serum:
Percentage of
Cells Killed
Effect of
Euglobulin:
Percentage of
Cells Killed
1
+++
+++
+++
++±
++
±
0
0
0
±
-
95%
90%
80%
80%
50%
95%
90%
60%
50%
30%
2
3
4
5
XVI: Showing That There Is Some Slight Variation in the Affinity of Different Agglutinating
Sera for the Particular Agglutinogens in the Red Blood Corpuscles of Certain Individual Rats
T ABLE
Red Blood
Corpuscles
of
1. R.S. Cream Hooded
2.
3.
4.
5.
6.
R.C. Fawn
L.S. Fawn
R.c. Black
L.S. Black
L.S. Black (Female)
Degree of
Agglutination
Anti-].R.S.
Serum
R.C. 19th
++±
++±
+++
++
++
±
Degree of
Degree of
Agglutination Agglutination
Anti-I.R.S.
Anti-RatSerum
Spleen
L.C. 18th
Serum B.C.
+
+
++
+±
+
±
+
±
+
±
±
±
Degree of
Agglutination
Anti-RatBlood Serum
L.C.C.H.
++±
++
++
±
0
±
the sera of normal rats, no agglutination occurs. It appears, therefore, that
these iso-agglutinins are related to homotoxins but not to heterotoxins.
(13) Six rats into which 1 c.c, of cell-free rat serum was repeatedly inoculated developed neither agglutinins nor chromatolysins in their sera (Table
IX).
( 14) Experiments previously recorded (5, 8) indicate that homocytotoxins exist mainly in the euglobulin fraction of sera containing them. Table
XV shows that this is not so in the case of iso-agglutinins.
( 15) The question of absorption has not yet been fully investigated, but it
is indicated by the experiments already carried out that agglutinins can be absorbed by either nucleated or non-nucleated cells, but that chromatolysins require nucleated cells for their absorption. It is doubtful, however, whether
chromatolysins can act in the entire absence of agglutinins, and further work
on this aspect is required. Among several difficulties in these experiments is
the marked lability of chromatolysins, and definite conclusions cannot be
drawn from a few experiments.
( 16) The experiments recorded in Table XVI suggest that there is some
slight difference in the agglutinogens present in the red blood corpuscles of
each particular agglutinin-sensitive rat, since there is some variation in their
408
THOMAS LUMSDEN
individual affinity for particular agglutinating sera. The variation is not very
great, however, and a serum which completely agglutinates the red blood
corpuscles of one, agglutinates those of all agglutinin-sensitive rats.
17) In order to discover whether a rat can manufacture chromatolysins
without at the same time producing haemagglutinins, a rat with agglutininsensitive corpuscles (R. C. F.) was immunised against rat spleen. As shown
in Table XVII, it produced a fairly high titre of chromatolytic homocytotoxins, but its serum contained no appreciable amount of agglutinins for red
blood corpuscles even of readily agglutinable variety. This observation and
those at the end of Section 11, threw light upon the general question of antibody formation, which is discussed below.
e
XVII: Showing That When a Rat with Agglutinin-sensitille Red Blood Corpuscles Is
Immunised against Rat Spleen Its Serum Contains Homotoxins but Not Haemagglutinins.
When an Unselected Normal Rat Is Immunised against Rat Blood the Reverse Occurs
TABLE
Nonsensitive
Ordinary
Rat
R.B.Cs.
Sera Drawn Three Days
After Last Immunising
Inoculation of
Tissue
Culture
of Spleen
(2 day)
Tissue
Culture
of I.R.S.
(2 day)
Rat spleen in agglutininsensitive rat (R.C.F.)
20
30
5
10
0
0
0
Rat spleen in ordinary
non-sensitive rat
45
55
5
5
+++
+±
0
0
0
+++
+++
0
Rat blood in ordinary rat
Agglutinin-sensitive Rat
R.B.Cs.
R.B.Cs.
R.S.C.H.
Fawn L.S.
(18) To determine whether the agglutinins found in the sera of rats immune to Jensen sarcoma and those immune to rat spleen were exclusively
haemagglutinins or were effective also upon nucleated cells, these sera were
applied to tissue cultures of rat spleen and of Jensen sarcoma. It was found
that agglutination of appropriate nucleated splenic and sarcomatous cells invariably occurred if they were not so rapidly killed as to prevent clumping.
It is evident that in the case of tissue cultures, agglutination of cells must take
some time, since it is only when the cells migrate within each other's orbit that
they are attracted to each other and adhere. If, therefore, we wish to observe agglutination of these living" cultured" cells, we must prevent rapid
death of the cells either by using a weak serum or by eliminating complement,
which is, of course, necessary for chromatolysis.
Another fact which supports the view that the iso-agglutinins we are considering are general cell agglutinins and not solely haemagglutinins is that they
are absorbed by nucleated cells (Jensen sarcoma) if anything more readily
than by red blood corpuscles. It is possible that absorption of this sort may
go on also if' corpore and may in part explain why, as long as an implanted
tumour is growing, agglutinins are absent from the serum of the rat concerned.
An observation which lends some support to this view was that in the case of
three rats in which a progressively growing tumour was excised, agglutinins,
previously absent, appeared in the serum, but, as might be expected, only in
small amount and for a short time (Table XVIII), since, if these rats had had
409
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
XVIII: Titre of Agglutinins at Various Dates in the Sera of Three Rats from Whuh
Progressively Growing Jensen Tumours Were Excised, Tested on Agglutinin-sensitive Rat Cells
TABLE
Dates
Before excision
1 day after excision
1 week after excision
10 days after excision
2 weeks after excision
Toxic effect on cultures of l.R.S.
(percentage of l.R.S. cells killed)
Rat R.C.
Rat B.C I •
Rat
B.C~.
0
0
0
0
0
0
±
±
±
±
±
±
0
0
0
2%
5%
5%
much capacity to form iso-agglutinins and homotoxins, the tumours implanted
into them would not have grown progressively.
( 19) The agglutinins are much more stable than the chromatolysins and
when once they attain a high titre they persist in an animal's serum for a considerable number of weeks; when, however, they are formed in an unsuitable
host, e.g. in a rat having some agglutinogen in its red blood corpuscles, they
may be in low titre and evanescent and so may be overlooked unless daily tests
are made (see Table XIII; Sections 9 and 11). The homocytotoxins are always extremely labile and are seldom if ever present in excess of the titre
necessary to achieve their purpose.
DISCUSSION
(1) The readily agglutinable red blood corpuscles investigated in the experiments recorded above supply a convenient means of studying natural and
acquired tumour immunity. The amount and nature of the agglutinogens
contained in any animal's cells appear to be determined by heredity and to remain unaltered throughout life. Genetic factors in relation to cancer have
been studied chiefly in America by Loeb, Little, Simpson, Bittner and many
others. It has long been known that strains of mice susceptible to, and others
resistant to, particular varieties of cancer could be bred, and in 1934 it was
noticed (see Fig. 5 in Lumsden, Macrae and Skipper, 5) that sera of rats immune to Jensen sarcoma contained iso-agglutinins capable of clumping homologous nucleated cells. Gorer (9), however, was the first to point out the occurrence of iso-haemagglutinins in this connection and to show that in his
pure strains of mice a particular variety of transplantable tumour grew progressively only in those animals whose red blood cells contained a particular
antigen, whereas if this antigen was absent, the tumours regressed and isohaemagglutinins appeared. My experiments were completed before reading
Gorer's paper and, in so far as Iso-haemagglutinins and agglutinogens are related to the fate of implanted tumours, there is, in principle, complete agreement between my results in unselected rats and those obtained by Gorer in
his pure bred mice.
(2) The Relation of Cell Agglutinins to Homocytotoxic Chromatolysins
(Homotoxins): The iso-agglutinins and the homotoxins usually run parallel
whether evoked by tumour implantations or by the inoculation of homologous
410
THOMAS LUMSDEN
TABLE
XIX: Showing Points of Difference Between Iso-agglutinins and Homoioxins
Effect produced
Amount in the euglobulin fraction
of the serum concerned. . . . . . . . . . . . . . . ..
Lability
Complement. . . . . . . . . . . . . . . . . . . . . . . . . . ..
Present in anti-rat-blood rat serum
Present in anti-rat-spleen serum
made in a rat having agglutinable
red blood corpuscles. . . . . . . . . . . . . . . . . . ..
Iso-agglutinins
Homocytotoxic
Chromatolysins
Agglutination, not
necessarily lethal
Nucleated cells killed,
chromatin lysed
Little if any
Very stable
None needed
Yes
Practically all
Very labile
Necessary for lysis
No
No
Yes
nucleated cells (Tables VIII-IX); yet they are apparently different antibodies, for under special circumstances each can be produced separately.
Thus the serum of rats immunised against rat blood cells may contain a high
titre of haemagglutinins though homotoxins are either absent, or present only
in such minimal amount as might be accounted for by the leucocytes in the
blood inoculum. Conversely, if rat spleen is used to immunise an agglutininsensitive rat, no haemagglutinins are formed, but homotoxins are demonstrable
in considerable amount (Table XVII). The chief points of difference between iso-agglutinins and chromatolysins are set out in Table XIX.
A hypothesis which fits all the known facts is that the agglutinins appear
in response to surface cytoplasmic antigens, the homotoxic chromatolysins to
nuclear antigens. It is, on this assumption, not surprising that the titre of
these two antibodies should run more or less parallel, since, under ordinary
conditions, when nucleated cells are injected the amount of surface and of
nuclear antigen would be comparable. The facts that red blood corpuscles
give rise only to agglutinins and that rat serum alone evokes neither agglutinins
nor chromatolysins, support this hypothesis. It is fortunate that iso-agglutinins, even when evoked by nucleated cells, do agglutinate red blood corpuscles,
since this supplies a much more ready means of demonstrating their existence
and their titre than the laborious and tedious tissue-culture tests by means of
which the existence of homotoxins was first discovered (5).
(3) On the Basis of Specificity and of Individual Variation in Response to
Antigens: In considering the specificity of the reactions between nucleated
cells and cytotoxic sera, too much attention may be focussed on the antibody
and too little on the antigen, though the latter is the more fundamentally important of the two factors. It is of value to reverse this point of view and,
taking the agglutination of cells as an example, consider specificity, primarily
as dependent upon antigenic structure and affinity.
When a rat is immunised again Jensen rat sarcoma, agglutinins appear in
the rat's serum which are capable of clumping the red blood corpuscles of
some rats, but not of others. This cytologic selectivity is not due solely to
the nature of the agglutinins in the antiserum, for they agglutinate not only
red blood corpuscles, but nucleated cells, normal as well as malignant. The
factor that mainly determines which cells shall be clumped, and which not, is
the presence or absence of the adequate agglutinogen in those cells. The titre
of agglutinins varies from one immune rat to another and from day to day;
411
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
TABLE
XX: Showing that in Rats Immunised against Homologous Cells the Titre of Agglutinins
Is Inversely Proportional to the Titre of Agglutinogens in the Red Blood
Corpuscles of the Rat Immunised *
Rat
Immunised
to
Serum of
Immunised
Rat plus
Agglutininsensitive
R.B.Cs.
Titre of
Agglutinins
Cells of
Immunised
Rat plus
Serum of
Anti-].RS.
Rat R. 19.
Titre of
Aggiutinogens
Cells of
Rat in
Own
Serum
Control
for
Cell
Suspension
Result of
].R.S.
Implant
++±
++±
+
±
±
±
0
0
Regressed
0
0
0
0
±
±
+
0
0
0
0
0
Regressed
+
±
0
Grew
Rat blood
R.C.L.1
L.C.C.H.2
I..C.I.. 3
R.C.C.H.4
R.C.B.5
L.C.B.6
0
0
Grew
Rat testis
R.C.C.l
RC.C. 2
RC.I.. 3
I..C.L.4
+++
++
±
±
0
* The anti-rat-blood rats 1 and 6 and the anti-testis rats 2 and 3 had ].R.S. implanted into
them; it will be seen that when the agglutinogen content of the rats' red blood corpuscles was
low the implant regressed; conversely when the agglutinogen content was high the ability to
form agglutinins and chromatolysins was low and the implant grew progressively.
the agglutinogen content of the cells is an inherited and extraordinarily stable
factor, determinable by selective breeding, but unalterable after birth by any
agency yet known.
We may regard the high agglutinability of the red blood corpuscles in certain rats as evidence that these cells contain an unusually large quantity of
agglutinogen, and for this reason one would expect them to be especially active as antigens, but another and an important deduction can be drawn,
namely, that a rat whose blood corpuscles or other cells contain much agglutinogen will not itself be capable of producing the corresponding agglutinins. This results from the fact that unless a substance is "alien" to the
animal it is inoculated into, it will not act as an antigen. If agglutinogen is
injected into a rat whose red blood corpuscles already contain that substance,
we are not supplying an " alien" antigen and no agglutinin will be formed.
Indeed from the experiments recorded in Section 11 (p. 405) and in Tables
XVII, XX, XXI, XXII, and XXIII, we may conclude that, inter alia, the
ability of an animal to produce agglutinin (antibody) varies inversely with the
agglutinogen (antigen) content of its cells.
It will be shown later that this law has an important bearing on natural
resistance to tumour implantation. See Tables XXI-XXIII.
The results obtained allow one to develop further the hypothesis that
412
THOMAS LUMSDEN
XXI: S/wwing That Rats in Which a Tumour is Growing Progressively N_ Contain
Agglutinins in Their Serum, While Their Red Blood Cells Contain an Unusually High
Titre of Agglutinogens for Unselected Rats of the London Lister Breed
TABLE
Serum
on Cells
R.S.C.H.
Titre of
Agglutinins
Cells
with Serum
of R.C. 19.
Titre of
Agglutinogens
0
0
0
0
0
0
0
0
0
0
0
±
±
+
+
+
±
0
0
0
0
0
0
0
0
0
0
0
12 X 5
0
+
0
10 X 5
5 X 2
0
0
+
+
0
0
0
0
+
+
0
0
0
Size of
Tumour in
Centimeters
Cells
with
Own
Serum
Nov. 22, '37
RC.
L.C.
B.C.
RS.
L.S.
8.5.
RC.L.S.
R.D.S.
L.D.S.
8.0.5.
R.C.L.D.S.
Nov. 8, '37
R.C.L.S.
6
7
7
7
5
5
6
6
7
7
6
X
X
X
X
X
X
X
X
X
X
X
1.5
1.6
1.5
1.6
2
1.5
1.7
1.6
1.7
1.5
1.2
Nov. IS, '37
R.S.
B.C.
Removed Tumours
RC.
8.C.(S).
8.C.(L).
+
±
±
±
+
±
cellular antigens vary in activity in proportion to the degree in which they are
" alien" to the animal into which they are inoculated. Thus heterologous
cells are a much stronger antigen than homologous cells and, as far as is
known, autologous cells (with the exception of spermatozoa and possibly of
cancer cells) do not act as antigens at all. Cancer cells, presumably because
they are more" alien" and invasive, are a more active antigen than any other
homologous type of cell. Next in order of potency are splenic fragments,
probably on account of the large number of wandering macrophages which
they contain. Definitely less effective are fixed tissue cells such as those of
the kidney or testis, and least active of all are red blood corpuscles.
Another consideration germane to tumour immunity is that within reasonable limits, the more active a celt is as an antigen and the more complicated its
antigenic pattern, the more sensitively and specifically it should react with the
antibody it evokes. For this reason antisera produced by inoculation of cancer cells have a degree of specificity against malignant cells quite apart from
specific" anti-cancer bodies" the existence of which has been suggested as the
result of previous experiments (7).2
2 It is difficult to demonstrate unexceptionably the existence of truly specific anticancer bodies
because they are always accompanied either by homotoxins or by increased" heterotoxins. Two
facts, however, indicate that they do exist. (1) It is shown in a former paper (7) that the serum
of a J. R. S.-immune rat is much more toxic than normal rat serum to tissue cultures of mouse
cancer. This is not so in the case of cultures of mouse spleen. In those experiments heterotoxins
are not increased, and since homotoxins which are present are not toxic to heterologous tissues, the
facts strongly suggest the existence of specific anticancer bodies in the J. R. S.-immune rat serum.
(2) The second fact emerged during the experiments recorded in this paper. It appears to be pos-
413
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
TABLE
XXII: Showing Tha; the Ability to Form Iso-agglutinins Varies from Rat to Rat and That
It Is in Inverse Proportion to the Agglutinogen Content of the IndividuaJ Rat's
Red Blood Cells: J.R.S. Non-Takes and Regressions
L.C.R.S.
R.S.
R.C.L.S.
R.C.L.D.S.
B.S.
R.D.S.
B.D.S.
L.S.
L.C.R.S.
L.D.S.
B.D.S.
L.C.-
Serum on Cells
of R.S.C.H.
Agglutininsensitive Rat
Titre of
Agglutinins
Cells with
Serum of
R.C.19
Anti- J.R.S. Rat
Titre of
Agglutinogens
Control
Cells on
Own Serum,
For Comparison
in Estimating
Low Titres
+++
+++
+++
+++
++±
++
+±
±
±
±
±
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
±
0
±
-±
0
0
±
±
0
0
- In this rat the tumour grew for a long time, but ultimately disappeared.' Possibly it had
a small titre of agglutinin at some time which was missed as the serum was not examined regularly.
(4) The Relation of These Experiments to the Study of Tumour Immunity: It has already been shown (7) that:
(i) The serum of each animal normally contains antibodies (heterotoxins)
lethal to nucleated cells of every foreign species which has been treated, but
not to homologous cells.
(ii) If the living nucleated cells from one animal are injected into another
animal, cytotoxins appear in the serum of the latter, lethal to the cells used as
an antigen.
(a) If the cells inoculated as an antigen are from a heterologous species,
not only are the non-species specific heterotoxins increased in titre, but antispecies bodies are produced in large amount. These anti-species bodies are
toxic to the cells of the species or group from which the antigen was taken, but
to no other variety of foreign cells, i.e. they are species-specific.
(b) If the cells inoculated as an antigen are homologous they evoke antibodies specifically toxic to the wandering cells (cancer cells and macrophages)
of animals of their own species or group = homocytotoxins or homotoxins,
These antibodies are chromatolytic, but do not lyse red blood corpuscles or
damage tissue cultures of fixed tissues (e.g. heart or kidney). The inoculated
sible to predict in wbich rats a J. R. S. implant will grow and in which it will regress only by using
the serum of a rat immunised against malignant cells (J. R. S.) in the estimation of the agglutinogens on which the prophecies are based. The sera of rats immunised against rat spleen or testis
fail to give selective results. This suggests that it is because the J. R. S.-immune rat serum contains anticancer bodies that it reacts with and can be used to estimate the particular agglutinogen
whose presence or absence determines the fate of tumour implantation in the rat whose red cells
are being tested.
414
THOMAS LUMSDEN
animal's own wandering cells are " protected" in some unknown way against
these homotoxins and are unharmed."
( 5) At least one variety of autocytoxin exists. The serum of a male rat,
mouse or guinea-pig is toxic to its own spermatozoa.
The protocols in this paper show that cell agglutinins are formed in association with the cytotoxic chromatolysins just mentioned and that the former
can be used as an approximate index of the latter. Both from the point of
view of confirmation and of extension of previous experiments, this is an advance of practical value, since the simple and rapid agglutination tests make it
possible to investigate even unlikely possibilities with negligible loss of time.
In every essential direction these agglutination experiments confirm the more
laborious and difficult, if more direct and conclusive, tissue culture tests.
Thus the demonstration by Lumsden and Phelps that, during regression of an
implanted tumour, chromatolysins can invariably be detected in varying
quantity in the serum of the rat concerned (see Table XIV) can be confirmed
(since the titre of isoagglutinins and homotoxins run parallel) easily and
cogently by demonstrating that:
(a) If a progressively growing tumour results from an implantation of
cancer, isoagglutinins are never demonstrable in the serum of the rat concerned
(Tables X-XIII).
(b) When spontaneous regression occurs, isoagglutinins can invariably be
demonstrated if frequent tests are carried out (Tables X-XIII).
(c) Occasionally while in a rat a tumour is still enlarging, isoagglutinins
begin to appear in its serum. If this happens the tumour always ceases to
grow and ultimately disappears (Table X, no. 3; XI, no. 4).
These facts suggest that chromatolysins and their associated agglutinins
are closely related to the mechanism by which an animal resists the invasion
of its tissues by cancer cells implanted into it and that they are, in part at
least, the basis of natural resistance.
It appears that an animal's natural resistance is directly proportional to its
capability to produce agglutinins and the more important chromatolysins, and
so is inversely proportional to the amount of agglutinogens in its body cells, of
which those in its red blood corpuscles act as an indicator. If this be so, from
estimating these agglutinogens by means of the serum of a rat immune to
Jensen sarcoma we should be able to predict with reasonable accuracy which
rats, out of any series into which tumour cells are about to be implanted, will
develop a progressively growing tumour and in which spontaneous regression
will take place.
Table XXIII shows with how much accuracy this prognostication can be
achieved, but the test is a delicate one and in each experiment the criterion
varies with activity of the agglutinating serum used and with the virulence of
S Basis of Protection: Since the above was written, it has dawned upon me that the interdependence and yet segregation of antigen and antibody supply a ready, now almost a self-evident,
explanation of the protection of an animal's own cells from antibodies made by it-autologous cells
contain none of the adequate antigen. That is why the animal was capable of forming the antibody. If, for example, the cells of a J. R. S.-implanted animal contain no agglutinogen (antigen),
it will be competent to form agglutinins and will destroy the implant. Its serum will agglutinate
the cells of other non-resistant rats (since they contain agglutinogen) but will have no effect upon
its own cells because they contain no antigen.
415
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
TABLE XXIII: Showing the Degree of Accuracy with Which It is Possible to Predict the Outcome
of J.R.S. Transplantationfrom Estimate of Agglutinogens in R.B.Cs.*
Rats
RB.Cs.
Rats
R.B.Cs.
+
Serum
Rat
of
Prophecy
R.C.19
Titre of
Agglutinogens
Result
Serum of
Rat +
R.B.Cs.
of
RS.C.H.
Agglutinins
+
Serum
Rat
of
Prophecy
R.C. 19
Titre of
Agglutinogens
3
±
+
±
4
0
S
11
±
±
±
±
±
±
±
Grow
Right
0
35
12
0
Regress
Right
36
13
0
Regress
Right
37
14
0
Regress
Right
++±
+++
±
38
15
Grow
Right
0
39
Grow
Right
0
17
±
±
±
Grow?
Right
18
0
Regress
Right
19
0
Regress
Right
20
±
±
±
±
±
Grow?
Right
Grow?
Wrong
Grow?
Right
0
46
Grow?
Wrong
±
47
Regress?
Wrong
0
48
1
2
6
7
8
9
10
16
21
22
23
24
Grow
25
Right
0
Grow
Wrong
+
26
Grow?
Right
0
27
Regress
Right
+++
28
Grow?
Right
0
29
Grow?
Right
0
30
Grow
Right
0
31
±
±
±
±
±
±
±
±
±
±
Grow
Serum of
Rat
R.B.Cs.
of
R.S.C.H.
Agglutinins
+
Result
Right
0
Grow
Right
0
Grow
Right
0
Regress
Wrong
0
Grow
Right
0
Grow
Wrong
Regress
Right
Grow
Wrong
Grow
Wrong
Regress
Right
0
Regress
Right
±
++±
+
±
±
+±
±
Grow
Right
0
~
Grow
Wrong
+
Grow
Right
0
Regress
Wrong
0
40
±
±
±
Regress
Wrong
0
0
41
0
Regress
Right
+±
±
42
0
Regress
Right
43
0
Regress
Right
++
+±
++
0
44
Right
0
45
+±
:!:
±
±
±
Grow
±
Regress
Right
+±
Grow
Right
0
Grow
Right
0
Grow
Right
0
Grow
Right
0
32
Grow?
Wrong
±
33
Grow?
Right
0
34
* The rats in this table were tested in 4 consecutive batches of 12 rats each (November and
December 1937). Rats 1 to 36 were black hooded white London rats, rats 37 to 48 were, in order,
2 cream hooded, 1 agouti hooded, 4 fawn, and 5 black rats.
The criterion varies in each experiment with, among other factors, the age and breed of the
rats, the titre of cell agglutinins in the serum of the ].R.S.-immune rat used, and with the virulence
of the tumour cells implanted. Hence even when the conditions are made as nearly comparable
as possible, complete success in prognosis cannot be expected. One striking fact emerges from
this and similar experiments. It is that when the titre of agglutinogens is nil (subminimal)
the tumour implant invariably regresses and prophecy in this instance is 100 per cent correct.
the tumour cells implanted, among other factors. With sufficient experience
and sensitivity, however, the prognosis seems to be accurate enough to indicate
that the principles upon which it is based cannot be far from the truth. A
416
THOMAS LUMSDEN
striking fact is that whenever the titre of agglutinogens is nil, regression invariably takes place.
It would be difficult to find a more perfect example of simple and balanced
mechanism than that responsible for natural resistance to invasion of the body
by alien cells. When on account of the low antigen content and consequent
insensitivity of an animal's cells a high titre of antibody is required, this animal has a proportionally high ability to produce it. How delicate this compensatory mechanism is can be appreciated from a careful study of Tables
XX-XXIII.
It seems. unlikely that this highly organised and regulated mechanism
should be merely fortuitous and purposeless; yet we cannot conceive that it
has been evoked (by gradual survival of the fittest) in response to circumstances (implantation of tissues and so on) which could hardly arise naturally,
and which have occurred artificially only for so short a space as forty or fifty
years. It is more reasonable to suppose that this mechanism of natural resistance is related to the prevention of the invasion of one tissue by another
within the limits of an individual body, and that investigation of this mechanism may throw light on the basis of the differentiation of tissues, and on Nature's method (quite possibly often successful) of preventing spontaneous
cancer.
The experiments recorded above refer to implanted tumours alone, and the
conclusions drawn from them are by no means necessarily applicable to the
spontaneous tumours or the lower animals or of man. Yet in the study of
tumour immunity, as we pass from the heterologous to the homologous, and
finally to autologous, we find that Nature's method of opposing invasion is
comparable for the first two; heterotoxins and antispecies bodies invariably
deal successfully with heterologous implantations; agglutinins and homotoxins
often succeed in repelling the invasion of homologous cells. It may be that a
similar though more refined and elusive mechanism exists which controls the
invasion even of autologous cells, for example the penetration of the chorionic
villi into the uterine wall. In other words, invading cells may be opposed by
similar means whether they are attempting to invade a heterologous, a homologous, or an autologous tissue.
SUMMARY AND CONCLUSIONS
( 1) The agglutinability of the red blood corpuscles of normal rats varies
widely. High agglutinin sensitivity is peculiar to certain individual rats. It
is hereditary and constant, but in the absence of selective breeding it is not an
invariable characteristic of any particular strain of rat.
(2) There are no blood groups in rats and they do not produce isohaemolysins demonstrable in vitro.
(3) The sera of normal untreated rats never contain iso-agglutinins, nor
do the sera of rats immunised against heterologous tissues such as mouse or
guinea-pig spleen.
(4) All rats immunised against homologous normal or malignant nucleated
cells develop iso-agglutinins and homotoxic chromatolysins in their sera, but
they vary much individually in their capacity to produce these antibodies.
AGGLUTINATION TESTS IN STUDY OF TUMOUR IMMUNITY
417
(5) Iso-agglutinins can best be demonstrated by applying them to readily
agglutinable rat red cells, i.e. red cells containing a high titre of agglutinogens.
The cells of ordinary unselected rats are unsuitable for this purpose.
(6) The activity of any cell as an antigen, and the degree of its reaction
with its specific antibody, vary directly with its antigen content. Conversely,
the ability of any particular animal (e.g. rat) to produce antibodies (e.g. isoagglutinins) is in inverse proportion to the antigen (e.g. agglutinogen) content
of its own cells.
(7) When evoked either by the regression of an implanted tumour, or by
inoculation of homologous nucleated cells, the titres of iso-agglutlnins and of
iso-chromatolysins run closely parallel.
(8) The more" alien" and" invasive" the cells used as antigens are, the
higher the titre of antibodies they evoke. Thus heterologous are more
powerful antigens than homologous cells, and autologous cells (except spermatozoa and possibly cancer cells) are non-antigenic. Of homologous nucleated
cells, cancer cells have been found to be the most powerful antigens; next
come tissues with many wandering macrophages, e.g, spleen. Fixed tissue
cells, e.g, testis or kidney, are less effective in the production of both agglutinins and of chromatolysins. Red blood cells generally evoke Iso-agglutinins,
but do not give rise to iso-chromatolysins, while blood serum, free from cells,
evokes neither of the cytotoxins named.
(9) The sera of rats in which a Jensen sarcoma is growing progressively
never agglutinate even readilyagglutinable rat red blood cells. The sera of
rats in which a tumour is regressing, or in which an implantation has failed to
grow, always contain iso-agglutinins and chromatolysins at some period.
( 10) The natural resistance of any rat to tumour implantation (J. R. S.)
is directly proportional to its capacity to produce agglutinins and chromatolysins and is inversely proportional to the amount of corresponding agglutinogen in its body cells, of which those in its blood corpuscles act as an approximate index. It follows that, by estimating these agglutinogens by means of
the strongly agglutinating serum of a rat immune to Jensen sarcoma, it is possible to select certain rats in which a tumour graft is sure to regress, and with
somewhat less accuracy others in which it will grow progressively.
NOTE: The writer is grateful to the British Empire Cancer Campaign for its constant
and generous support, financial and otherwise. He thanks also his colleagues, both clinical
and scientific, at the London Hospital for help in every way possible.
REFERENCES
1. BORDET, J.: Ann. de l'Inst. Pasteur 12: 688, 1898.
2. EHRLICH, P., AND MORGENROTH, ].: Bed. klin. Wchnschr. 37: 453, 1900.
3. TODD, C., AND WHITE, R. G.: Proc. Roy. Soc., ser. B, 82: 416, 1910; 84: 255, 1911.
]. Hyg. 10: 185, 1910.
4. TODD, C.: Proc, Roy. Soc., ser. B, 106: 20, 1930.
5. LUMSDEN, T., MACRAE, T. F., AND SKIPPER, E.: J. Path. & Bact. 39: 595, 1934; 40: 418,
1935.
6. LUMSDEN, T., AND PHELPS, H. J.: Am. J. Cancer 29: 517, 1937.
7. LUMSDEN, T.: Am. J. Cancer 31: 430, 1937.
8. LUMSDEN, T.: Am. ]. Cancer 15: 563, 1931.
9. GORER, P. A.: J. Path. & Bact. 44: 691, 1937.