Cellular Immunity Against Rous Sarcomas of Chickens. Preferential Reactivity Against Autochthonous Target Cells as Determined by Lymphocyte ...
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Proc. Nat. Acad. Sci. USA
Vol. 71, No. 9, pp. 3565-3569, September 1974
Cellular Immunity Against Rous Sarcomas of Chickens. Preferential
Reactivity Against Autochthonous Target Cells as Determined
by Lymphocyte Adherence and Cytotoxicity Tests In Vitro
(neoplasm/tumor/Rous sarcoma virus)
M. A. WAINBERG, Y. MARKSON, D. W. WEISS, AND F. DOLJANSKI
The Lautenberg Center for General and Tumor Immunology, and the Department of Experimental Medicine and Cancer Research,
Hebrew University Medical School-Hadassah Hospital, Jerusalem
Communicated by George Klein, May 20, 1974
ABSTRACT Spleen cells from random-bred chickens competence for recognizing the autochthonous than allo-
bearing Rous sarcomas were commonly more reactive geneic neoplastic targets, and a frequently superior ability to
against the neoplastic target cells'in autochthonous than inflict injury on the autochthonous tumor cells. If preferential
in allogeneic interactions in vitro. This difference was
observed both in cytotoxic assays (hCr release from labeled autochthonous recognition will prove to be a general phe-
target cells) and in an immunoadherence test measuring nomenon in tumor immunology, resort to autochthonous target
attachment of $"Cr-labeled splenocytes to Rous sarcoma cells for the assessment of resistance in animals and man in
cells. Specific splenocyte reactivity was not observed with vitro will have to be considered a condition for accuracy and
normal embryonic chicken fibroblasts, 3T3 cells, or em-
bryonic mouse C3H fibro'blasts. The immunoadherence relevance.
technique required only 2 hr to perform, and revealed a
more consistent superiority of autochthonous recognition MATERIALS AND METHODS
of Rous sarcoma cells than the cytotoxicity assay. Ex- Virus. The Schmidt-Ruppin strain of Rous sarcoma virus
periments in which both procedures were used simul-
taneously with identical cell populations yielded similar was obtained from Dr. A. Kohn, Israel Institute for Biological
resiilts, indicating that splenocyte adherence may be a Research, Ness Ziona, and propagated in cultures of embry-
precursor of and/or concomitant to target cell damage onic chicken fibroblasts by the method of Temin and Rubin
and that individual-specific tumor antigencity may play (5). Supernatant fluids containing infective virus were col-
a part in cellular immunity against Rouis sarcomas.
lected from cultures almost fully transformed, clarified by
The host-tumor relationship of chickens bearing Rous sar- lQw-speed centrifugation, and frozen at - 70° until use.
comas has a number of advantages as a model pertinent to Chickens and Tumor Induction. Randomly bred, 6- to 10-
neoplasia in man. The tumor host is an outbred, nonlabora- week-old White Leghorn chickens, both males and females,
tory adapted animal, and the virus that induces the neoplastic were injected in their right wing webs with about 106 focus-
transformation is one of a family of agents whose members are forming units of the virus. About 3-4 weeks later, by which
oncogenic in nature as well as in the laboratory, and active time sizeable tumors had developed in most instances, the
in a broad range of species in addition to the chicken (1). neoplasms were removed surgically from some of the animals.
Cellular and humoral immunity in this system is well estab- Three days after surgery, the birds were killed and their
lished both in vito and in vitro (2, 3), and the chicken lends spleens harvested. Other tumor-bearing chickens, which had
itself uniquely to the separation of lymphoid cell populations not been subjected to surgery, were killed at the same time,
of diverse origin, and thereby to a differential definition of and their spleens taken. Age-matched normal chickens served
immunological capacity (4). as normal spleen donors.
The purpose of the present investigation was to compare
splenocyte-target cell interaction in both autochthonous and Cultivation of Rous Sarcoma (RS) Cells in Tissue Culture.
allogeneic confrontations between Rous sarcoma cells and Tumor tissue was cut into 2-mm3 pieces, which were then
effector cells, by use of splenocytes sensitized during the subjected to 3-4 cycles of trypsinization of 20 min each at
course of the host's experience with a primary, actively grow- 370 in calcium and magnesium-free phosphate-buffered saline
ing tumor-a situation reflecting the only type of interaction containing 0.25% (w/v) trypsin (1: 250 Difco Laboratories,
that can be studied in man. Detroit, Mich.). Tumor cells obtained from the second
This interaction was evaluated by a newly developed through fourth trypsinizations were suspended in standard
quantitative immunoadherence test, and by the cytotoxicity medium [Medium 199 (Grand Island Biological Co., Grand
assay based on the liberation of labeled chromium from target Island, N.Y.) supplemented, with 5-10% inactivated calf
cells. serum (In Vitro, Jerusalem), 10% tryptose phosphate broth
The findings presented here show that splenocytes from (Difco Laboratories), 200 units/ml of penicillin, 200 jg/ml
primary Rous sarcoma hosts have a consistently superior of streptomycin, and 15-25 ml/liter of 5% NaHCOs]. The
cells were washed by centrifugation, pooled, counted, and
Abbreviations: RS, Rous sarcoma; standard medium, medium seeded.
199 supplemented with serum, tryptose phosphate, and anti-
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biotics; insulin medium, standard medium in which serum is Other Cell Types. Normal chicken embryo cells (CEC) were
replaced by insulin. secondary cultures (2- to 3-days-old) derived from 9- to 11-
35653566 Immunology: Wainberg et al. Proc. Nat. Acad. Sci. U$A 7i (i974)
mined. The radioactivity of the adherent splenocyte popula-
tion was determined by trypsinizing the target cells with the
15- adherent splenocytes and counting as above. -Levels of spleno-
cyte adherence were calculated as the percentage of the label
remaining with the adherent cells by the following equation:
w % Adherence
0
zio
w
cpm of adherent splenocytes X 100
a:
I total cpm of adherent + nonadherent splenocytes
Microcytotocy Assays were performed by incubating
61Cr4abeled RS cells with suspensions of splenocytes at 370,
and measuring the amounts of label released. For this pur-
pose, 6 X 10' freshly obtained tumor cells in 0.2 ml of standard
medium were seeded into each microwell of a tissue culture
microtest plate (Tissue culture microtest 1480, Nunc A.S.,
V 0 60 120 180 270 Denmark), and incubated at 370 in a 5% CO-containing
MINUTES OF INCUBATION humidified atmosphere. The medium was changed daily;
FIG. 1. Adherence of normal and sensitized 51Cr-labeled after 3 days the cells of each well were labeled with 4-5 pCi of
splenocytes to Rous sarcoma cells. 0, Normal splenocytes; SICr in 20 pl of Hank's balanced salt solution at 370 for 1 hr.
O, sensitized splenocytes to allogeneic RS cells; *, 'sensitized The wells were washed four times with Haniks' balanced salt
splenocytes to autochthonous RS cells.
solution and splenocytes were suspended in 0.2 ml of standard
medium adde at 'a splenocyte: target cell ratio of 50:1 or
day-old embryos and grown in standard medium. Normal 100:1. In most experiments, splenocytes and target cells of
mouse fibroblasts were tertiary cultures of 19- to 20-day-old the same origin were used simultaneously for bQth the im-
embryos of the 03H strain. Another source of mouse embryo munoadherence and cytotoxicity tests.
cells was the 3T3 line (6), kindly provided by Dr. M. Inbar After various times of incubation at 370, culture fluids
of the Weizmann Institute, Rehovoth. from each well were removed with an Eppendorf pipette,
transferred to disposable 0.5-ml -plastic test tubes, and counted
Splenocytes. Spleen cell suspensions were prepared by within larger plastic tubes for released radioactivity, All ex-
teasing the spleens of normal and tumor-bearing chickens in perimpents were performed with at least four replicate samples.
sterile insulin medium [standard medium in which serum was Qytotoxic activity was expressed as the percentage of total
replaced by 40 units of insulin (Burroughs-Wellcome & Co., label released. Specific cytotoxicity was expressed as the per.-
London) per liter]. After large fragmpents had settled out, the centage of 61Cr released by sensitized splenocytes, according
cells were washed three times in the same medium by low- to'the'following equation:
speed centrifugation. Cell numbers were determined by count-
ing in a hemocytometer. More than 90% of the cells thus % Specific 5Cr release
obtained were always viable, as judged by trypan blue ex- cpm released cpm
clusion. Splenocytes from normal chickens will be referred to in presence of released in
as "normal splenocytes," and those from tumor-bearing sensitized presence of
chickens as "sensitized splenocytes." splenocytes normal splenocytes
X 100
Quantitative Immunoadherence Assays were performed by cpm initially incorporated*
measuring levels of adherence of 5"Cr-labeled splenocytes to Statistical Analysis. The significance of differences between
RS target cells. For this purpose, 106 viable tumor cells in 2 sensitized autochthonQus and allogeneic splenocytes, and be-
ml of standard medium were seeded into 35-mm plastic cul- tween each of these and normal splenocytes, was analyzed
ture dishes (Nunc. A.S., Denmark). The medium was changed by the one-tailed paired t test, for both the adherence and
every 24 hr and replaced after 72 hr with insulin medium. microcytotoxicity tests in the over-all comparisons.
The cells werelabeled by the method of Wigzell (7) by in-
cubating 108 cells in 2 ml of insulin medium containing 70 RESULTS
;Ci of PCr (The Radiochemical Center, Amersham, En- Adherence of Normal and Sensitized Splenocytes to RS Target
gland) for 30 min at 37°. They were then washed four times by Cells. Three-day-old newly established cultures of RS cells,
centrifugation and suspended in insulin medium to the de- consisting of both characteristic round cells and fibroblast-
sired concentration. Radioactivity was determined in an like cells (8), were incubated in parallel with autochthonous
Autogamma spectrophotometer (Packard). Aliquots (0.1 ml splenocytes, with splenocytes from another tumor-bearing
each) of the labeled splenocyte suspension were then added chicken, and with splenocytes from a normal control bird.
to target cells monolayers 'at a splenocyte: RS cell ratio' of After 1 hr at 370, a striking clustering of splenocytes on the
50:1 or 100:1. The culture plates were incubated for various target monolayer was consistently evident in the autochtho-
periods of time at 370 in a 5%0 CO2 humidified incubator, nous combination, and to a lesser extent in the 4llogeneic one;
after which the 'supernatant fluids containing nonadherent
splenocytes were collected. The cultures were then washed * Total incorporated radioactivity was determined by counting
three times writh 2 ml of insulin medium to remove additional the supernatant fluid .and cells removed by trypsinization from
nonadherent cells. The splenocytes in the combined super- control wells (four in each experiment) containing target cells
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natants were sedimented and their radioactivity was deter- only.Proc. Nat. Acad. Sci. USA 71 (1974) Cellular Immunity to Rous Sarcomas 3567
allogeneic clustering was always appreciably more marked
than that seen with normal splenocytes. In order to quantitate
this phenomenon, we labeled splenocytes with 5"Cr and the
radioactivity of the adherent spleen cell population was de-
termined. Fig. 1 shows the results of a typical experiment.
Splenocyte adherence increased with time during all three
types of interaction, before leveling off after 2-3 hr. It is also
evident that autochthonous splenocyte reactivity was more
pronounced than allogeneic reactivity, which in turn was
better than that evinced by the control spleen cells. These Lu
:t
differences were observed in every one of 16 consecutive LU
2
experiments, and were cumulatively significant (P < 0.0005;
one-tailed paired t test). cq
In order to rule out the possibility that the greater ad-
herence effected by autochthonous splenocytes arose as a
consequence of surgical removal of part of the tumor 3 days
before the effector cells were harvested, "criss-cross" experi-
ments were performed, in which target cells were prepared
from each of two donors on which we had performed opera-
tions. When the splenocytes of each donor were tested against TumorA Tumor B
both the authochthonous and allogeneic tumor cells in com- Target cells
parison with splenocytes from a third tumor-bearing animal FIG. 2. Adherence of sensitized splenocytes to Rous sarcoma
on which we had not operated, we found that the highest cells after 3 hr of interaction at splenocyte-target cell ratios of
levels of adherence again occurred in the autochthonous inter- 100:1 ("criss-cross" experiment). _, Splenocytes from tumor-
actions. The results of one such experiment are shown in Fig. bearing chicken A; , splenocytes from tumor-bearing chicken
2. Two further criss-cross experiments provided identical B; 1:.:1, splenocytes from tumor-bearing chicken [Q C; I,
findings. splenocytes from control.
To test the specificity of the adherence phenomenon, we
incubated splenocytes from both normal and tumor-bearing ential reactivity of sensitized splenocytes against autochtho-
donors with different types of normal cells in culture. Fig. 3 nous target cells was again evident in both systems. It is seen
shows that, with respect to normal chicken embryo cells, that the sensitized splenocytes showing the greatest immuno-
cells of the 3T3 line, and C3H mouse fibroblasts, the adherence adherence activity were generally also the most potent in the
capacities of both types of splenocyte populations did not cytotoxicity tests. It thus appears that adherence of spleno-
differ. cytes to RS cells, which takes place within 2 hours after con-
tact, may be concomitant to, and possibly a forerunner of,
Cytotoxic Effect of Normal and Sensitized Splenocytes on severe target cell injury, as indicated by specific 51Cr release.
RS Cells. Normal spleen cells did not effect a release of 51Cr
above spontaneous release levels under the conditions of these DISCUSSION
experiments. A systematic comparison was undertaken of the specific re-
The results of eight consecutive trials analyzing the cyto- activities of spleen cells from chickens with Rous sarcomas
toxic capacity of sensitized splenocytes against autochthonous against newly established cultures of the autochthonous and
and allogeneic target cells are summarized in Table 1. Inter-
action with sensitized autochthonous splenocytes caused sig-
nificant cell damage in 9 of 11 cases, whereas such effect was
seen only in 9 of 17 cases for sensitized allogeneic splenocytes.
The autochthonous interaction was significantly more dam- 30-
aging than the allogeneic one in 6 of the 11 cases, whereas in no
instance was the reverse true. The cytotoxic activity of sensi-
tized splenocytes against both the autochthonous and the
allogeneic target cells was first significant after 6 hr of inter-
action. Lu.
LU
o20
In order to ascertain the specificity of RS target cell killing
by splenocytes from tumor-bearing donors, 61Cr-release ex-
periments were also performed with monolayers of normal
chicken embryo cells. Table 1 (Experiments 6 and 8) shows
that there was no significant difference in the amounts of
tTA Fhri
10
70
11l
51Cr released from monolayers of normal chicken embryo TumorA Norrnal chick C3H 3 T3
cells interacting with normal or sensitized splenocytes. target embryo
TARGET CELLS
Comparison of Results from Immunoadherence and Cell- FIG. 3. Adherence of sensitized and normal splenocytes to
Mediated Cytotoxicity Tests with Identical Cell Populations. Rous sarcoma and other target cells after 3 hr of interaction at
Fig. 4 summarizes the results of six of the eight experiments splenocyte target ratio of 100:1. _, Splenocytes from tumor-
depicted in Table 1 in which both cytotoxicity and immuno- bearing chicken A; 1, splenocytes from tumor-bearing chicken
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adherence tests were conducted simultaneously. The prefer- B; 2, normal splenocytes.3568 Immunology: Wainberg et al. Proc. Nat. Acad. Sci. USA 71 (1974)
TABLE 1. Cytotoxic actiity of splenocytes from normal and tumor-bearing chickn agaimt Row sarcoma target cells and normal
chicken embryo cells
% "'Cr Release when the effector cells were:
Sensitized,
Splenocyte/ Sensitized, autochthonous
target cell Target allogeneic to to the target
Exp. ratio cells Normal the target cells Pn* cells Pn Pat
1 100:1 Tumor a 12.45 14.22 0.05
25.59 0.01
Tumor b 11.39 15.42 0.05 38.57 0.01 0.01
Tumor c 10.39 32.14 0.01 29.23 0.01 NS
2 50:1 Tumor d 7.60 10.07 0.02 17.91 0.01 0.01
3 50:1 Tumor e 23.9 21.3 NS
24.3 NS
Tumor f 17.3 19.7 NS 27.4) 0.01 0.01
4 50:1 Tumor g 17.7 18.6 NS 37.3 0.01 0.01
25.2 0.01
Tumor h 13.2 26.0. 0.01 41.8 0.01 0.01
Tumor i 17.3 17.7 NS 19.4 NS NS
19.4 NS
5 50:1 Tumor j 18.2 21.5 NS 28.3 0.05 0.05
6 50:1 Tumor k 30.1 34.1 0.05 34.9 0.05 NS
CEC 39.7 44.4 NS
46.3 NS
7 100:1 Tumor 1 13.3 15.8 NS 12.3 NS NS
8 50:1 Tumorm 22.63 26.16 0.01 25.73 0.01 NS
CEC 16.7 14.3 NS
14.6 NS
CEC, Normal chicken embryo cells; NS, not significant.
* Probability of significant difference from normal splenocytes.
t Probability of significant difference from allogeneic splenocytes.
of allogeneic Rous tumor cells. As indicated by the adherence against autochthonous as compared with allogeneic target
of splenocytes to the neoplastic target cells, and by spleno- cells. Moreover, these workers could detect lymphocyte-
cyte-mediated cytotoxicity, a greater degree of reactivity was mediated cytotoxicity only with effector cells from birds with
frequently observed in autochthonous interactions. The regressing tumors, whereas we found such activity in spleno-
superiority of autochthonous recognition and cytotoxic ca- cytes from chickens with progressively developing sarcomas.
pacity was highly significant despite the considerable varia- These differences in results could be due in part to the use by
tion from host to host in the extent of splenocyte immune Hayami et al. of birds from a small, closed breeding colony,
adherence and cytotoxic action against both autochthonous i.e., a population of tumor hosts with a narrowed range of
and allogeneic targets. These observations confirm previous of variant individual characteristics; in addition, the quails
reports of cellular immune manifestations in chickens against were challenged simultaneously with two tumors, a circum-
Rous sarcomas (3), and they point to the existence of a host- stance that could lead more readily to a state of specific
specific dimension of immunological ability. immunological unresponsiveness.
Gelderblom et al. (9) and Kurth and Bauer (10) demon- The adherence and clustering of sensitized lymphoid cells
strated that at least two distinct cell-surface antigens are on allogeneic normal target cells have been extensively studied
associated with chicken cells transformed by Rous sarcoma (13, 14) and have also been described for a number of tumor
virus: a group-specific antigen of avian leukosis virus expressed target cell systems (15-18). However, the adherence phenome-
only on the cell surface of the transformed cell, and a virus non has not previously been applied to systematic and quanti-
subgroup-specific antigen located on the envelope of virus tative analysis of tumor immunity. The quantitative immuno-
particles budding from the plasma membrane. Our findings adherence technique here described appears to constitute a
of an additional individual-specific aspect of host reactivity reliable and very sensitive means of revealing the presence of
to RS cells could reflect either qualitative or quantitative effector cells specifically directed at tumor-associated anti-
variations from tumor to tumor in the expression of one or gens. Thus, preferential autochthonous reactivity was evident
another of these Rous-associated antigens, or the occurrence in 100% of the comparisons by the immunoadherence test,
of still other antigens, in some way unique for each tumor, but in only about half the instances by the criterion of cell-
and perhaps not directly associated with the oncogenic agent. mediated cytotoxicity. If the assumption is made that firm
Other workers have also reported the appearance of protective adherence to the target cells is a first stage of cell-cell inter-
antigens specific to individual tumors, superimposed on the actions, but not one necessarily followed by severe damage to
crossreactive antigens associated with a common oncogenic the targets (19), immunoadherence assays could provide a
virus (11). quantitative picture of the baseline of immune recognition.
Hayami et al. (12), working with Rous sarcomas in quails, The extent of adherence of normal splenocytes to RS cells
did not detect differences in cellular immunity directed was generally high and varied considerably from donor to
Downloaded by guest on August 8, 2021Proc. Nat. Acad. Sci. USA 71 (1974) Cellular Immunity to Rous Sarcomas 3569
cytotoxicity reactions, and the possibility remains open that
armed, as well as "activated," macrophages (23) and B
iu 60- lymphocytes with specific cytophilic antibodies, as well as
specifically reactive T lymphocytes, participated. Attempts
40-
to clarify the role of T and B lymphocytes in the Rous sarcoma
0q 24 5 system have been recently made with bursectomized chickens
(24, 25), and it seems that both T and B cells are involved in
0-
cellular anti-tumor activities.
Q
60-
This work was supported by Contracts NIH 71-2127 and NIH
70-2208 from the National Cancer Institute, and by research
L1J 40-
grants from the Leukemia Foundation, Inc., Concern Founda-
tion, Inc., The Lautenberg Endowment Fund, and Mr. and Mrs.
.-'20-
Laurence Tisch. M.A.W. is a postdoctoral fellow of the European
Molecular Biology Organization.
0 2 3 4 5 6
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EXPERIMENT NUMBER 2. Jonsson, N. & Sjogren, H. 0. (1965) J. Exp. Med. 122, 403-
FIG. 4. Summary of six experiments comparing immuno- 421.
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against Rous sarcoma cells. _, Autochthonous interaction;
2437.
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