241
THE EFFECT OF LYMPH-NODE CELLS FROM SPECIFICALLY
SENSITISED MICE, ON THE GROWTH OF THE BP8 ASCITES
TUMOUR " IN VIVO " AND " IN VITRO "
G. A. W. ROOK AND D. B. CATER*
From the Department of Pathology, Univer8ity of Cambridge
Received for publication November 22, 1967
EHRLICH (1909) first suggested that immunological factors might play a part in
the host's resistance to tumours. Since then much research effort has been wasted
by the failure to use isogeneic animals for transplantation experiments and in fruitless search for tumour specific antibodies. These latter are difficult to demonstrate
and, according to recent reviews (Allison, 1967; Alexander and Fairlie, 1967), may
not be the essential factor in the host's resistance to the tumour. If, on the other
hand, this is related to the delayed type of hypersensitivity and to homograft
rejection then lymphocytes will play an important role. Indeed, immunity to
tumour induction in some experimental systems can be transferred by lymphocytes or peritoneal macrophages in the same way as homograft immunity (Old,
Boyse and Lilly, 1963). Tumour growth is also inhibited if tumour cells are incubated with lymphoid cells from immunised animals before injection into syngeneic
hosts (Klein, Sj6gren, Klein and Hellstrom, 1960) or with spleen cells (Mikulska,
Smith and Alexander, 1966).
These experiments could be interpreted in two ways:
(a) a cytotoxic action by immunised lymphocytes against tumour cells
(b) the injected immunised cells " instruct " the hosts reticulo-endothelial
system to react against the tumour.
There is considerable evidence to support both these views, which in any case
are not mutually exclusive.
Cytotoxic effects have been reported by a number of workers (Rosenau and
Moon, 1961; Wilson, 1965; Brondz, 1964; M6ller, 1965) who incubated target-cell
monolayers with lymphoid cells from immunised animals. The target cells
included normal or neoplastic cells from allogeneic strains, or, when tumourspecific antigens were strong, neoplastic cells from syngeneic animals.
In favour of the second mechanism are the experiments of Najarian and Feldman
(1961) who transferred delayed skin sensitivity by labelled cells and found that
subsequent testing with the antigen gave a lesion in which only a tiny percentage of
the reacting mononuclear cells were labelled. Also, experiments in which the
lymphoid cells were not injected with the tumour cells but were given intravenously,
support the second mechanism (Delorme and Alexander, 1964), particularly in
those cases where the lymphoid cells had come from a different species and would
not be expected to survive for long in the host (Hassan and Stuart, 1963; Alexander,
Delorme and Hall, 1966).
*
Gibb Fellow of the British Empire Cancer Campaign for Research.
G. A. W. ROOK AND D. B. CATER
242
Cater and Waldman (1967) showed that BP8 ascites tumour cells were inhibited in vivo by lymphocytes from C57B1 mice which had been immunised with
BP8 cells in Freund's complete adjuvant. This tumour was induced by benzpyrene in C3H mice and has strong H-2K histocompatibility antigens which are not
present in C57B1 mice (Haughton, 1964). The in vivo tests were carried out by
injection of the tumour and lymphocytes i.p. into C3H/C57B1 Fl hybrids or C3H
mice.
It was therefore decided to investigate the mechanism of this inhibition using
both in vivo and in vitro techniques.
MATERIALS AND METHODS
Passage of BP8 tuMour cells
BP8 cells were taken from storage at -180°, rapidly thawed at 370, centrifuged and resuspended in TC 199 to remove the Spooner medium, and 5 X 104 viable cells were injected
into C3H mice i.p. Seven days later the mice were killed by cervical dislocation, 1 ml. of
heparinised Hanks' solution (5 units/ml.) was injected i.p. and the ascitic fluid was removed
under sterile precautions using a plastic syringe without a needle. Siliconed glassware or
sterile plastic vessels were used for all manipulations of cells. The tumour was passaged by
injecting 5 x 104 viable cells in 0- 1 ml. i.p. into C3H mice.
Immunisation of C57Bl mice
The antigen consisted of 40 x 106 BP8 cells/ml. killed by freeze/thawing 3 times plus an
equal volume of Freund's complete adjuvant (Difco). The mixture was emulsified by passing
it repeatedly from one syringe to another through 3 cm. of fine polythene tubing. The first
dose was 4 x 106 cells plus 0.1 ml. of Freund's adjuvant per mouse injected into 4 sites. The
mice were injected at weekly intervals and from the fourth injection onwards 1 X 106 cells
plus 0 025 ml. of Freund's adjuvant was used.
Preparation
of lymph-node cell suspensions
C57B1 mice, which had received at least 3 injections, were killed by cervical dislocation 7
days after the last injection. The lymph nodes were removed with sterile precautions,
minced with fine scissors, suspended in TC 199, and filtered through 3 thicknesses of muslin
gauze.
(Pipetting the suspension was avoided; it broke up any remaining fragments but
greatly reduced the viability
of the cells.)
Tissue Culture in Rose Chambers
The Rose Chamber is a " sandwich " of non-toxic rubber between 2 coverslips, all clamped
There is a cavity in the rubber which leaves a 2 ml.
chamber, bounded above and below by coverslips, which can be filled by passing hypodermic
needles through the rubber.
The coverslips were polished, placed in stainless steel racks, immersed in Pyroneg non-toxic
detergent at 50° for 40 min. and then washed in many changes of distilled water and dried in
the racks. Additional washing with HC1 or ethanol did not improve the cultures. The TCV,
white, non-toxic rubber (supplied by Esco (rubber) Ltd.) was washed by prolonged boiling in
several changes of distilled water. (It was slightly toxic for BP8 cells when new, but boiling
removed the toxic agent.) The chambers were then assembled and autoclaved.
BP8 cells/ml. (2 x 105), harvested as described above, were incubated at 370 in TC 199
plus 10 per cent foetal-calf serum (Glaxo Laboratories). The medium was changed every
24 hr.
together by 2 nickel-plated brass plates.
Method for assessing attachment of lymnph-node cells to tumour cells in Rose Chambers
The medium was drained from 24 hr. BP8 cultures and was replaced by medium containing
4-5 x 106 lymph-node cells/ml. (i.e. about 20 per tumour cell). The chambers were orientated
so that the lymphocytes fell by gravity on to the tumour cells and were then incubated for
24 hr. Then the chambers were inverted, a third of the medium was removed, and they were
SENSITISED LYMPH-NODE CELLS AND ASCITES TUMOUR
243
gently rocked so that the bubble travelled over the cell layer. This removed dead tumour
cells and any unattached lymph-node cells. This technique was standardised as far as
possible, but in any case healthy tumour cells did not come off the glass unless the shaking was
violent or prolonged. The chambers were filled with fresh medium, their numbers were
covered to eliminate subjective error, and differential counts were made, under phase contrast,
of 20 random fields.
RESULTS
Expt. 1.-The effect of sensitised lymph-node cells given i.v. on the growth of BP8
tumour in vivo
Lymph-node cells 5-7 x 106 (from C57B1 mice which had received 6 immunising
injections of BP8 cells plus Freund's complete adjuvant) were given i.v. to each of 6
C3H/C57B1 F1 hybrid mice. Four control mice from the same batches were given
no lymphoid cells. All the mice were challenged with 5 X 104 BP8 cells i.p.
TABLE I.-Expt. 1: C3H/C57Bl F1 Hybrid Mice were Protected by Intravenous
Injection of 5-7 x 106 Lymph-node Cells, from " Immunised" C57Bl Mice,
Against Fatal Challenge with 5 x 104 BP8 Tumour Cells i.p.
Treatment
Protected
Sex
Survival time in days
Male
58
Male
296*
Female
296*
Female
296*
Female
113
Female
296*
Male
Unprotected
18
Male
18
Female
18
Female
23
* Experiment terminated at 296 days, these mice were healthy and at autopsy no abnormalities
w^-ere seen.
All the controls died within 18-23 days, with ascites tumour only. Two of the
protected mice died after prolonged survival (at 59 and 113 days). Solid tumours
wNvere found infiltrating the omentum and 1 mouse had a tumour in its left kidney, a
section of which is shown in Fig. 1. There is a pronounced mononuclear-cell
reaction round the tumour. No ascites tumour was found in the protected mice.
The 4 surviving protected mice were killed at 296 days. They were all healthy and
no abnormalities were found at autopsy.
Expt. 2.-The effect of sensitised lymph-node cells given i.v. on the growth of BP8
tumour in previously irradiated animals
This experiment was designed to show whether the sensitised lymph-node cells
would have the protective effect, demonstrated in Expt. 1, even if the immunological responses of the recipient mice were damaged by 450 r total body irradiation.
The procedure was identical to Expt. 1 except that there were 2 groups of protected
animals one of which was irradiated before injection. The controls were also
irradiated.
The result of Expt. 1 was confirmed, but no protection was observed in the
irradiated animals. Their spleen, lymph-nodes and thymus were all small. There
244
G. A. W. ROOK AND D. B. CATER
TABLEII.-Expt. 2: The Effect of Sensitised Lymph-node CellsGiven Intravenosly,
on the Growth of BP8 Tumour in Animals Given 450 r Total Body Radiation
(220 KVp X-rays)
Treatment
All mice were challenged with BP8
5 x 104 cells i.p.
"Protected"
5-7 x 106 lymph-node cells i.v.
"Protected" + 450 r total body
radiation
5-7 x 106 lymph-node cells i.v.
Sex
.
.
.
"Unprotected " + 450 r total body
radiation
.
Survival time in days
M
M
F
F
.
.
.
241*
241*
241*
37
M
M
M
F
F
.
.
.
.
.
17
21
13
16
18
M
M
F
F
.
.
.
.
15
16
16
16
* Experiment terminated at 241 days, these mice were healthy and at autopsy no abnormalities
were seen.
was a great reduction of all the cells in the spleen seen in section. There was also a
tendency of the tumour to invade, producing numerous small nodules to which
there was no mononuclear-cell reaction (Fig. 2). Such invasion was not found in
non-irradiated animals, except in " protected " mice which had died after prolonged survival. As mentioned above in these cases a few large tumours were
found with evidence of lymphocytic reaction.
This experiment indicated that the protective effect given by the sensitised
lymphocytes was found only when the hosts immune responses were active.
Possibly the injected sensitised lymph-node cells contained a " transfer factor"
which enabled the host's cells to react against the tumour.
EXPLANATION OF PLATES
FIG. 1. Part of a solid BP8 tumour in the left kidney of a C3H/C57 F1 hybrid mouse which had
been protected by i.v. injection of 5-7 x 106 lymph-node cells from immunised C57B1 mice
against fatal challenge with BP8 tumour cells i.p. It survived 58 days (Table I). There is a
conspicuous mononuclear reaction. H. and E. x 150.
FIG. 2. A solid BP8 tumour infiltrating the muscle of the abdominal wall of a C3H/C57 F1
hybrid mouse which had received 450 r total body radiation (Table II). It survived 18
days. There is no cellular reaction against the tumour. H. and E. x 150.
FIG. 3. BP8 cells, 24 hr. culture. H. and E. x 180.
FIG. 4. BP8 cells, 24 hr. culture. Phase contrast. x 210.
FIG. 5.-Lymph-node cells from immunised C57B1 mice attached to BP8 cells in living culture.
Phase contrast. x 300.
FIG. 6. BP8 cell culture after incubation for 24 hr. with lymph-node cells from control C57B1
mice. Only a few lymphocytes are attached to the BP8 cells many of which have long
processes. H. and E. x 150.
FIG. 7. BP8 cell culture after incubation for 24 hr. with lymph-node cells from immunised
C57B1 mice. Note that far more lymph-node cells remain attached to the BP8 cells and that
these are degenerating compared with Fig. 6. H. and E. x 150.
BRITISH JOURNAL
VOl. XLIX, NO. 3.
OF EXPERIMENTAL PATHOLOGY.
,4'
:71:
L;
s_
.01
.'
_w
I
}.
l
,
iA.
va L. ,.
0.
.w.:
0
401. A.1.111.1.60,1
Rook and Cater.
BRITI8H JOURNAL
OF
EXPERIMENTAL PATHOLOGY.
VOl. XLIX, NO. 3.
Rook and Cater.
SENSITISED LYMPH-NODE CELLS AND ASCITES TUMOUR
245
TABLE III.-Expt. 3: The Effect of Sensitised Lymph-node Cells, Irradiated with
500 r and Given i.v. on the Growth of BP8 Tumour in C3H/C57Bl Hybrid Mice
Treatment
All mice were challenged with BP8
5 x 104 cells i.p.
"Protected " with:
7-5 x 106 lymph-node cells i.v.
.
.
"Protected " with:
7.5 x 106 lymph-node cells, irradiated 500 r, i.v.
.
"Protected " with:
7-5 x 106 lymph-node cells i.p.
"Protected" + 450 r total body radiation
7-5 x 106 lymph-node cells i.v.
"Unprotected"
.
.
Sex
M
M
M
M
F
M
M
M
F
F
F
M
M
F
F
F
F
M
F
F
F
M
M
F
F
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Survival time in days
109*
109*
109*
109*
109*
109*
109*
109*
109*
109*
109*
109*
109*
109*
109*
109*
109*
12
109*
13
14
18
18
18
18
* Experiment terminated at 109 days, mice healthy, at autopsy no abnormalities seen.
Expt. 3.-The effect of irradiating the sensitised lymph-node cells and then giving
them i.v. on the growth of BP8 tumour in vivo
In this experiment 4 groups of hybrid mice were protected with 7X5 X 106
lymph-node cells from immunised C57B1 mice. In the first group shown in Table
III the hybrid mice were protected by intravenous injection against challenge by
5 X 104 BP8 cells given i.p. This proved that the lymph-node cells were effective
and confirmed the results obtained with the similar groups in Expts. 1 and 2. In
the second group an aliquot from the same pool of lymph-node cells was irradiated
with 500 r and then injected i.v. This small dose of irradiation would be sufficient
to prevent the cells dividing for some hours but would be unlikely to kill a high
proportion or damage antigens. This group showed long-term survival. The
third group was protected with lymph-node cells i.p. and showed long-term survival
as would be expected from the results of Cater and Waldmann (1967). The fourth
group was a repeat of Expt. 2 and showed that after the hybrid mice had been given
450 r that 3 out of 4 were not protected, but that one mouse was protected by
7.5 x 106 lymph-node cells given i.v. The fifth group were an unprotected control
group and were all found dead on the nineteenth day.
This experiment is consistent with the hypothesis that lymph-node cells from
the immunised C57 mice contain a "transfer factor ", or in some way hand on
information, which enables the host cells to react against the tumour.
246
G. A. W. ROOK AND D. B. CATER
Expt. 4.-To investigate the cytotoxic effect of lymph-node cells from immunised
C57Bl mice on BP8 tumour cells in vitro
Ten Rose Chamber cultures of BP8 cells (Figs. 3 and 4) were prepared and,
after incubation for 24 hr., were divided at random into 2 groups. Five chambers
were filled with medium containing 5 x 106 lymph-node cells/ml. from C57B1 mice
which had been immunised (4 injections of BP8 cells plus Freund's complete
adjuvant). The other chambers were filled with a similar suspension of lymphnode cells prepared from non-immunised C57B1 mice. After a further 24 hr. of
incubation the medium was changed, the dead cells removed and the remaining
cells counted, as described under Methods.
Statistical analysis of the results shown in Table IV by Student's " t " test
showed that the total number of tumour cells was significantly lower in the
chambers containing the immunised lymph-node cells (t = 5-46, P < 0 1 per cent).
The number of lymph-node cells attached to each tumour was significantly higher
as shown by the difference in the lymphocyte/tumour cell ratios (t = 2-343, P <
0-1 per cent).
TABLE IV.-Expt. 4: The Cytotoxic Effect on BP8 Cells In Vitro of Lymph-node
Cells Prepared from Immunised C57BI Mice Compared with that of Lymph-node
Cells from Control C57Bl mice,
Total cell counts of 20 random fields
Chambers treated with lymph-node
cells from immunised C57 mice
Lymph-node
BP8 cells
cells
164
304
67
123
96
231
132
116
88
129
Mean 109-4 ± 17-2
180-6 ± 37*4
Mean lymph-node cell/BP8 cell ratio
1-688 + 0-252
Chambers treated with lymph-node
cells from control C57 mice
BP8 cells
337
341
280
285
189
286-4 ± 27-4
Lymph-node
cells
308
465
356
252
141
304-4 4- 53-8
1-035 ± 0119
This experiment was repeated 4 times, and the 2 effects observed were
consistent.
Thus when the lymph-node cells came from injected mice the ratio of lymphocytes to tumour cells was always greater, as seen in Fig. 7 compared with Fig. 6.
The total number of tumour cells was also lower-note that in Fig. 6 there are not
only fewer lymphocytes attached to the tumour cells but the BP8 cells show more
processes, which is a sign of healthy cells, also in Fig. 7 some of the BP8 cells show
obvious signs of degeneration. It therefore seemed that the sensitised lymphoid
cells were capable of specific attachment to the target cells (Figs. 5 and 7) and also
produced a cytotoxic effect. It was decided to investigate the relationship between
these 2 effects.
Expt. 5.-To test the effect in vitro of antibody to BP8 cells on the specific attachment
of lymph-node cells prepared from immunised C57Bl mice
Serum from C57B1 mice which had received 5 injections of BP8 cells in Freund's
complete adjuvant was prepared from the heart blood of ether-anaesthetised mice.
SENSITISED LYMPH-NODE CELLS AND ASCITES TUMOUR
9d4 7
Any complement activity was inactivated by heating to 560 for 30 min., and the
serum was then titrated against BP8 cells in the presence of rabbit complement.
A cytolytic effect was observed up to a dilution of 1/400.
Four out of 8 BP8 cell cultures were incubated for 30 min. at 370 with a 1/8
dilution of this antiserum. Then all the cultures were washed with TC 199 and
refilled with culture medium containing 5 x 106 lymph-node cells/ml. from immunised C57B1 mice. After 24 hr. the medium was changed, loose cells were washed
away, and viable cell counts were made.
TABLE V.-Expt. 5: Effect of Pretreating BP8 Cells with Antibody on the Specific
Attachment to them of Lymph-node Cells from Immunised C57B1 Mice
Total cell counts of 20 random fields
BP8 cells pretreated with Mouse AntiBP8 serum J. Then 5 x 106 lymphnode cells/ml. from immunised C57B1
BP8 cells received no pre-treatment.
Then 5 x 106 lymph-node cells/ml.
from immunised C57B1 mice
mice
,
A
A
BP8 cells
Lymph-node cells
204
360
243
300
279
359
301
229
Mean 238-75 ± 15-65 330 ± 17-03
Mean lymph-node cell/BP8 cell ratio
1-40 + 0-123
,
A
sA
BP8 cells
322
270
281
284
289-25 + 11-3
Lymph-node cells
172
159
214
203
187 i 12-9
0-65 + 0-053
Statistical analysis of the results shown in Table V indicated that more of the
antibody-coated tumour cells had died (t = 2-614, P about 4 per cent); and that
antibody had increased the number of lymphocytes attached to the tumour cells
(lymph-node cell/tumour cell ratios, t = 5-612, P between 1 per cent and 0-1 per
cent).
In retrospect, another control should have been included in this experiment,
consisting of BP8 cells pretreated with anti-BP8 serum, washed and then covered
with lymph-node cells from non-immunised C57 mice. This would have tested the
possibility that BP8 cells were damaged by the antiserum and then became sticky,
simply because they were damaged. Subject to the possibility of such a nonspecific synergism it did appear that the antibody had acted synergistically with
the immunised lymph-node cells. This raised the possibility that the lymph-node
cells were producing antibody in the Rose chambers.
Expt. 6.-To test for the in vitro production of antibody in the presence of BP8
antigens, by lymph-node cells from C57B1 mice which had received 5 injections of
BP8 cells in Freund's complete adjuvant
Five BP8-cell cultures containing 2 x 105 cells/ml. were prepared in Rose
chambers. After 24 hr. incubation the medium was replaced with a suspension of
5 x 106 lymph-node cells/ml. prepared from C57B1 mice which had received 5
immunising injections. The chambers were turned upside down so that the lymphnode cells were not in contact with the tumour cells and incubated for a further
24 hr. Then the lymph-node cells were washed out and the BP8 cultures were
rinsed twice in complement-fixation diluent (supplied by Difco).
248
G. A. W. ROOK AND D. B. CATER
Rabbit complement was then titrated against other BP8 cells in the presence
and absence of anti-BP8 serum, so that a dilution could be chosen, at which there
was full complement activity, but no " anti-mouse " effect. The BP8 cells in the
Rose chambers prepared as above were then exposed to the complement while the
chambers were watched under phase-contrast microscopy.
Result.-No lysis of tumour cells was observed.
Expt. 7. To investigate the effect of bringing together by centrifugation BP8 cells and
lymph-node cells from immunised C57Bl mice
BP8 cells were mixed in various proportions with lymph-node cells prepared
from C57B1 mice which had received 5 injections of BP8 cells plus Freund's complete adjuvant. These mixtures were centrifuged at 2000 r.p.m. for 2 min. in
siliconed tubes. The supernatants were then removed by inverting the tubes and
the pellets of cells were gently resuspended in the drops which remained. These
were placed on siliconed slides and examined by phase-contrast microscopy.
Controls were done using lymph-node cells from non-immunised mice.
Result.-It was not possible to demonstrate by this technique any specific
attachment of sensitised lymph-node cells to BP8 tumour cells.
DISCUSSION
As mentioned in the introduction there are at least 2 possible mechanisms for
the transfer of tumour resistance by sensitised lymph-node cells.
(1) The injected sensitised lymph-node cells may themselves destroy the
tumour cells, with or without undergoing mitosis within the host animal.
(2) The lymph-node cells may transfer to the host's immunological systems, the
ability to react quickly to the tumour.
These suggestions are clearly not mutually exclusive. One cell-type could be
capable of both mechanisms, or there could be 2 different active cell-types within
the heterogeneous population of a lymph-node cell suspension.
Few attempts have been made in vivo to distinguish between these mechanisms
using tumours, but much work has been done on homograft rejection and delayed
hypersensitivity. Such work merits discussion here, since whether or not tumourspecific antigens can provoke these reactions, the histocompatibility antigens
which tumour cells also carry, are known to do so. It has been shown that BP8
cells carry the strong H-2K antigens (Haughton, 1964).
In vivo work does not support direct action by transferred sensitised lymphocytes on target cells. If delayed hypersensitivity is passively transferred by cells
labelled with tritiated thymidine, less than 10 per cent of the mononuclear cells
infiltrating a specifically-induced lesion will be labelled (Najarian and Feldman,
1961). It is therefore interesting that delayed hypersensitivity has been transferred in humans, with cell-free extracts (Lawrence, 1956 and 1959).
Work with tumours leads to similar conclusions. Lymphoid cells from the
lymph of specifically-immunised sheep were found to inhibit the growth of primary
sarcomata in rats (Alexander, Delorme and Hall, 1966). This effect was prolonged, yet " graft versus host " disease was not observed and it seemed unlikely
that sheep lymphocytes could survive in rats for more than a few days. These
workers subsequently obtained a similar effect with an impure RNA extract of the
sheep lymphocytes (Alexander, Delorme, Hall, and Hamilton, 1967).
SENSITISED LYMPH-NODE CELLS AND ASCITES TUMOUR
249
The C57B1 lymph-node cells used in our experiments were injected intravenously into C3H/C57B1 F1 hybrid mice, but no graft versus host disease was
observed. They gave excellent inhibition of tumour growth, unless the animals
had previously received 450 r total body irradiation, which caused marked degeneration of the spleen and lymph-nodes, but was not fatal. It also appears that these
lymph-node cells gave better protection when injected intravenously, than when
injected i.p. with the tumour cells.
The ratio of injected lymph-node cells to BP8 cells was only 100/1 and the
tumour could be expected to undergo rapid mitosis. Thus unless the lymph-node
cells contained a very high percentage of competent cells, which " homed " very
efficiently on to the tumour, a direct action could not have caused its complete
disappearance. It is important to note that the lymph-node cells may have been
sensitised to the tumour-specific antigens, or to H-2K antigens, which are also
present in the tissues of the C3H/C57B1 F1 hybrid mice. In the latter case they
could attack any tissue.
The evidence presented here suggests that in vivo the sensitised lymph-node
cells did not act by " homing " on the tumour cells and destroying them, but the in
vitro experiments showed that our cell preparations were in fact capable of specific
attachment to, and destruction of BP8-cell cultures. Many workers have reported
similar effects. The necessity for contact between the lymphocytes and the target
cells was established by the use of millipore chambers (Rosenau, 1963). The
specificity of the reaction has also been established with both normal and neoplastic target cells (Brondz, 1964; Rosenau and Moon, 1961) and quantitative
studies have suggested that one lymphocyte can kill one target cell (Wilson, 1965)
Other workers, however, have shown that non-sensitised lymphocytes can kill
target cells in the presence of phytohaemagglutinin. The cells must be genetically
dissimilar but the lymphocyte need not be genetically competent to react against
the target cell. Thus an F1 hybrid lymphocyte can kill a parental cell in the
presence of phytohaemagglutinin (Holm, Perlmann and Werner, 1964; Moller and
Moller, 1965). In view of the fact that P.H.A. causes transformation of lymphocytes, it could be argued that in this situation it by-passes the need for sensitisation, thus giving the impression that the reaction is non-specific. (Coulson and
Chalmers, 1964.)
Moller also stated that in his experimental system, antibody to the target cells
blocked the attachment of the lymphocytes. However in our experiments, and in
those of Govaerts (1964), antibody and lymph-node cells acted synergistically.
Certain in vivo investigations may be relevant to these in vitro results. Inhibition
of tumour cells in mice by a combination of antibody and lymphocytes has been
reported (Batchelor, Boyse, Gorer, 1960). In their experiments lymphocytes
alone were ineffective, while serum alone actually stimulated the tumour.
However Berne (1965), working with BP8 antiserum showed inhibition of
tumour growth followed by enhancement in C57B1/DBA hybrid mice. Indeed this
appears to be the usual effect, but a detailed discussion of the mechanism of tumour
enhancement would be out of place here. There is no evidence to show that the
effect Moller observed in vitro is the in vivo mechanism. The 2 systems may have
nothing whatever to do with one another.
The explanation for the contradictory views on the effect of antibody on the
specific attachment of lymphocytes to target cells may lie in the possibility that the
cellular and humoral responses are determined by different antigenic configura-
250
G. A. W. ROOK AND D. B. CATER
tions. Thus if the antibody combined with a site which did not bear a close
structural relationship to the site for which the lymphocyte was specific, it would
not block the lymphocyte's attachment. In other cases blockage could occur.
In conclusion we suggest that lymph-node cells from specifically-sensitised
C57B1 mice can kill BP8 tumour cells in culture, but probably give in vivo protection to C3H/C57B1 F1 hybrid mice by acting as a transfer factor.
SUMMARY
C57B1 mice were given subcutaneous injections of BP8 tumour cells emulsified
in Freund's complete adjuvant. Cell suspensions were made from the lymph
nodes of these animals.
These cells, when injected i.v. made C3H/C57B1 F1 hybrid mice resistant to i.p.
injections of BP8 cells. This resistance was not seen if the host mice were previously irradiated with 450 r.
The sensitised lymph-node cells also had cytotoxic effects on BP8 cell monolayers in vitro. This effect was accentuated if the tumour cells were first incubated
in anti-BP8 serum, taken from the C57B1 mice.
REFERENCES
ALEXANDER, P., DELORME, E. J. AND HALL, J. G.-(1966) Lancet, i, 1186.
ALEXANDER, P., DELORME, E. J., HALL, J. G. AND HAMILTON, L. D. G.-(1967) Nature,
Lond., 213, 569.
ALEXANDER, P. AND FAIRLEE, G. H.-(1967) Br. med. Bull., 23, 86.
ALLISON, A. C.-(1967) Br. med. Bull., 23, page 60.
BATCHELOR, J. R., BOYSE, E. A. AND GORER, P. A.-(1960) Transplant. Bull, 26, 449.
BERNE, B. H.-(1965) Proc. Soc. exp. Biol. Med., 118, 228.
BRONDZ, B. D.-(1964) Folia biol., Praha, 10, 164.
CATER, D. B. AND WALDMAN, H.-(1967) Br. J. Cancer, 21, 124.
COULSON, A. S. AND CHALMERS, D. G.-(1964) Lancet, ii, 819.
DELORME, E. J. AND ALEXANDER, P.-(1964) Lancet, ii, 117.
EHRLICH, P.-(1909) Nederl. ijdsbhr. Geneesk. Amst, 1, 273.
GOVAERTS, A. J.-(1964) In 'Immunological Methods ', ed. Ackroyd, J. F. Oxford.
(Blackwell) pp. 224-237.
HASSAN, A. M. AND STUART, A. E.-(1963) Lancet, ii, 496.
HAUGHTON, G.-(1964) Transplantation, 2, 251.
HOLM, G., PERLMANN, P. AND WERNER, B.-(1964) Nature, Lond., 203, 841.
KLEIN, G., SJ6GREN, H. O., KLEIN, E. AND HELLSTROM, K. E.-(1960) CancerRes.,20,
1561.
LAWRENCE, H. S.-(1956) Bull. N. Y. Acad. Med., 32, 236.
LAWRENCE, H. S.-(1959) In 'Cellular and Humoral Aspects of the Hypersensitive
States', ed. Lawrence, H. S., New York (Harper). pp. 279-318.
MIKULSKA, Z. B., SMITH, C. AND ALEXANDER, P.-(1966) J. natn. Cancer Inst., 36, 29.
MOLLER, E.-(1965) J. exp. Med., 122, 11.
MOLLER, E. AND MOLLER, G.-(1965) In 'Isoantigens and cell interactions' (ed. Joy
Palm) Symposium Monograph 3. (Philadelphia) Wistar Institute.
NAJARIAN, J. S. AND FELDMAN, J. D.-(1961) J. exp. Med., 114, 779.
OLD, L. J., BOYSE, E. A. AND LILLY, F.-(1963) In 'Cell-bound Antibodies' ed. Amos, B.
Koprowski, H, Philadelphia. (Wistar Institute) p. 89.
ROSENAU, W.-(1963) In'Cell-bound antibodies' ed. Amos, B. Koprowski, H. Philadelphia (Wistar Institute), pp. 75-83.
ROSENAU, W. AND MOON, H. D.-(1961) J. nat. Cancer Inst., 27, 471.
WILSON, D. B.-(1965) J. exp. Med., 122, 143.