[CANCER RESEARCH 37, 1154-1158, April 1977)
Lymphoid Subpopulation Changes in Regional Lymph Nodes in
Squamous Head and Neck Cancer1
Andrew Saxon and John Portis
Department of Microbiology and Immunology/Immunobiology
California 90024
Group (A. 5.1 and Department of Pathology (J. P.), UCLA School of Medicine, Los Angeles,
SUMMARY
Lymph nodes from 10 normal patients and regional lymph
nodes (RLN) from 19 patients with squamous cancer of the
head and neck were evaluated as to their lymphoid subpop
ulations. In comparison to normal lymph nodes, RLN from
cancer patients demonstrated a marked increase in the
proportion of cells with membrane immunoglobulin, the
receptor for the third component of complement, and the
receptor for the Fc portion of immunoglobulin G. The in
creased Fc receptor cells were not Fc-beaning thymus-de
nived lymphocytes, inasmuch as they separated with the
non-sheep erythnocyte-lymphocyte rosette-forming popula
tion. The overall thymus-derived lymphocyte percentage in
RLN was proportionally decreased. A transition from the
normal lymph node composition to the altered lymphocyte
profile seen in RLN was demonstrated on moving from
distal lymph nodes to RLN within the lymphatic drainage of
a tumor. Lymph nodes involved with tumor also showed the
pattern of bursa equivalent cell population increases.
INTRODUCTION
RLN2 constitute an early line defense against infection (3,
21),
transplanted
tissues
(7,
22),
and
cancers
(30).
The
effect of the RLN on tumor growth is controversial. Fisher
and Fisher (10) and Alexander and Hall (1) have presented
evidence that these local LN are important in initiation and
maintenance of antitumor activity, while other investigators
(5, 11, 12, 14) have been unable to demonstrate either in
vitro antitumor activity by RLN cells or their importance in in
vivo tumor control.
Many advances in relating pathological tissue changes to
the nature of disease have followed the introduction of new
methodologies for characterizing cellular changes, e.g.,
histochemical stains and electron microscopy. Histological
changes in RLN have been studied for some time (6, 8, 25).
Recent developments in immunology permit the recogni
tion of various normal lymphoid subpopulations by surface
I
Research
supported
by
NIH
Grant
CA
12800
and
NIHTraining
Grant
Al
00431.
2 The abbreviations
used are: RLN, regional
lymph node(s);
LN, lymph
node(s); NLN, normal lymph node(s); MIg, membrane-bound immunoglobu
lin; T-ceIl, thymus-derived cell; D' N, distal lymph node(s); MEM HEPES,
minimal essential media buffered to pH 7.4 with 1.0 u N-2-hydroxyethylpiper
azine-N'-2-ethanesulfonic acid; PBL, peripheral blood lymphocytes; E-ro
sette, rosette formation between lymphocytes and untreated sheep RBC; Zy
C, complement coated zymosan particles; B-cell bursa equivalent cell; PBS,
0.15N phosphate-buffered
saline.
Received September 27, 1976; accepted December 29, 1976.
1154
receptor characteristics. Evaluation of RLN composition by
these immunological techniques is indicated to explore the
alterations on the lymphoid tissue closest to the neoplastic
growth. This paper analyzes the changes in lymphoid sub
population in LN under the stimulus of proximity to active
neoplastic tissue. In comparison to NLN, RLN from cancer
patients demonstrated a profound increase in the propor
tion of cells bearing MIg, the receptor for the 3rd compo
nent of complement (C'), and the receptor for the Fc portion
of lgG (Fc). Concomitantly, there was a decrease in the
percentage of T-cells in RLN.
MATERIALS AND METHODS
Patients. LN were obtained from 18 patients with squa
mous cancer ofthe head and neck who were undergoing LN
dissection as part of their surgical treatment. Patients had
received no prior radiation, chemotherapy, on immunother
apy except where specifically noted. No patient had evi
dence of distant metastases. Two patients with squamous
cancer of the head and neck who received radiation therapy
consisting of 4500 nads oven 5 weeks preopenatively sup
pliedradiatedLN. NLN were obtainedfrom patientsun
dergoing cosmetic surgery. The mean age for head and
neck patients was 60 ±9 years; for normals it was 42 ±12
years.
Lymphocytes. All LN studied were from the cervical or
axillary regions. Only LN greaten than 0.5 cm in diameter
and immediately draining the tumor were used as RLN. RLN
that were grossly or microscopically involved with tumor
were excluded, except where otherwise noted. DLN were
taken from the distant margins of the same surgical speci
mens. The LN were obtained fresh, placed in a 100- x 20-mm
Falcon petni dish with MEM HEPES, and then teased apart
using two 18-gauge needles. To remove debris, the result
ant suspension was passed through a 12-mI plastic syringe
with a 1-mI nylon wool plug. The cells were then washed 3
times in MEM HEPES and brought to a final concentration
of 107/ml. PBL were obtained by Ficoll-Hypaque separation
as previously reported (29). PBL from 7 patients were ob
tamed at least 1 month after surgery.
Rosette Procedures. T-cells were enumerated by E-ro
sette formation (18). Sheep RBC (Mission Labs, Rosemead,
Calif.) were washed 4 times in 0.15 M PBS, and resuspended
as a 1% suspension in fetal calf serum (Reheis Chemical
Co. , Chicago, Ill.) that had been heat inactivated at 56°for
30 mm and absorbed with 10% sheep RBC at both 4°and 37°
for 1 hr. Equal volumes of this 1% suspension and lympho
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RLN Populations in Cancer
cytes at 0.5 x i07/ml were mixed with 10- x 75-mm Pyrex
tube (Scientific Products, Irvine, Calif.) and incubated at 4°
for at least 1 hr. The pellet was then resuspended by gently
notating the tube on its long axis, 0.05 ml of crystal violet
stain was added, and an aliquot was counted at the hemo
cytometen chamber. Lymphocytes with 3 or more sheep
RBC were
counted
as rosettes.
Cells with a receptor for the Fc portion of IgG (Fc cells)
were enumerated by rosette formation between lympho
cytes and trypsinized sheep RBC coated with hypenimmune
rabbit anti-sheep RBC antibody (32). Trypsinization pro
vents the formation of lymphocyte E-rosettes. Twenty ml of
5% suspension of sheep RBC in MEM HEPES were incu
bated with 100 mg of lyophilized trypsin (Sigma Chemical
Co., St. Louis, Mo.) for 30 mm at 37°followed by 3 more
washes. The antibody-coated trypsinized sheep ABC were
then suspended in MEM HEPES to a final concentration of
10 x 107/ml. The nosetting and counting procedure was the
same as for E-nosettes.
Complement receptor-bearing cells (C' cells) were enu
menated by lymphocyte rosette formation with Zy-C (17).
Zymosan particles (Sigma) were swollen in boiling PBS for
30 mm, washed twice with PBS, and adjusted to 1 mg
zymosan per ml. Fresh human sena, 0.5 mI/mg zymosan,
were added, and the mixture was incubated with agitation
at 37°for 30 mm. The zymosan-complement complex was
washed 3 times in PBS, adjusted to a final concentration of
1 mg Zy-C per ml, aliquoted , and stored at —70°.
Upon
thawing, it was stored at 4°and used for 3 days. The lym
phocyte zymosan rosettes were formed and counted by
a procedure identical to that for E-nosettes except that the
mixture was not incubated at 37°and was shaken prior to
counting.
Immunofluorescence. Lymphocytes with MIg were Ia
beled by direct immunofluorescence (27). After incubation
to remove cytophilic IgG (19), between 0.5 and 0.1 x i0@
lymphocytes were incubated at 23°with a 1:10 dilution of
polyvalent goat anti-human immunoglobulin (Meloy Labo
ratonies, Springfield, Va.) in MEM HEPES with 0.01% azide.
The cells were then washed 3 times with cold MEM HEPES
plus 0.01% azide, mounted on a glass slide, and examined
with epi-illumination on a Leitz Orthoplan microscope. The
percentage of fluorescent cells was determined, and the
presence or absence of capping was noted. For removal of
any aggregated immunoglobulin that would bind to Fc me
ceptors, the antisera were originally ultracentnifuged at
45,000x g for90 mm, storedat4°,
and respunat10,000x g
for30 mm justpriorto use.
LN T- and B-Cell Separation. A fractionation of LN T- and
B-cells was affected by E-rosette separation on Ficoll-Hy
paque (13) except that the sheep RBC were pretreated with
2-aminoethylisothiounonium bromide hydrobmomide. This
modified technique yields highly purified T- and B-Iympho
cyte fractions (29).
Miscellaneous. Viability was tested by trypan blue dye
exclusion (0.40%). Esterase-positive cells (monocytes) were
counted by the a-naphthyl acetate method (31), while con
tamination with granulocytes was enumerated by Wright's
stain of cytocentnifuge-prepared slides. Phagocytic cells
were determined by ingestion of i-im latex beads (33) (Dow
Chemical Corp.,Midland,Mich.).Latexbeads were incu
bated with (0.1 to 0.2 x 10@)cells in MEM HEPES plus 25%
fetal calf serum for 60 mm at 37°and washed 3 times with
PBS; the percentage of cells with intracellular beads was
determined after Wright's stain of these cells.
RESULTS
RLN. The majority of cells from LN immediately draining a
primary cancer were T-cells (54.3%). However, this percent
age was significantly less than seen in NLN, or the patients'
or normals' PBL (p < 0.001) (Table 1). Along with this
decrease in percentage of T-cells was an increase in cells
carrying the C' receptor, MIg, and Fc receptor (p < 0.01),
the latter population having the largest proportional in
crease. There was no significant difference between the
percentage of these 3 populations (Fc, C', MIg; p > 0.05).
These changes were not due to systemic changes in circu
lating lymphocytes as the patients' PBL subpopulations
were unchanged from normal PBL. Since Fc receptors can
be expressed on B- on T-cells (2), T-cells were purified and
retested for the presence of Fc-positive cells. NLN T-cell
fractions showed 5.66 ± 2.65% (S.D.) Fc-positive cells
while 5.39 ±4.85% of RLN T-cells expressed an Fc recep
ton. Conclusive evidence that the T-ceII population with an
Fc receptor for lgG was not expanded will rest on simulta
neous double marker studies. The MIg-positive cells
showed capping of the fluorescent stain at a level equal to
that seen in PBL or NLN cells. There was no difference. Our
Table 1
Percentageof cells bearing sheep ABC receptor, Fc receptor, C', and MIg from normal
blood, normal
DLN'sNo.
lymph nodes, patients' blood, RLN's, and
ofsam-
ABCpIes
Sheep
C'MIgLymphoid
receptorcell
LNRLN
6.4NLN
2.1DLN
Fc
subpopulations
in
23
54 ±3.7°
25 ±6.6
10
74 ±5.6
7 ±3.8
12±5.112±3.6Lymphoid
6
68±8.3
12±7.2
20 ±4.025
9 ±3.510
±
±
NLNNLN
subpopulations in blood and
9±3.510±2.1Normal
10
74±5.6
7±3.8
blood
3.9Patient
10
65 ±6.1
17 ±5.3
12 ±4.414
±
2.9aMeanblood
±S.D.
7
64 ±5.2
19 ±6.0
11 ±4.912
±
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1155
A. Saxon and J. Portis
findings cannot be accounted for by erosion of the primary
tumor to a body surface with secondary bacterial invasion,
since this did not occur in 7 cases. Gmanulocytes, esterase
positive cells, and phagocytic cells comprised less than 1%
of each sample from RLN as they did for NLN. The lack of
phagocytic and esterase-positive cells was not due to loss
of these populations during LN disruption on filtration, inas
much as their number was not increased in the prefiltration
lymphoid suspension even when all cells were mechanically
freed from the Petni dish. Viability was 84.7 ±3.4%.
To determine whether these changes in LN composition
were related to proximity to the primary tumor or could be
found diftusely in the LN, LN from different anatomical
locations were studied from 2 patients undergoing radical
neck dissection for squamous cancer of the head and neck.
The cellular composition was determined for an immedi
ately draining node greater than 0.5 cm in diameter (RLN),
several small nodes (<0.3 cm) from the same anatomical
area, and a distant lymph node (Table 2). In one case, the
location of the tumor (laryngeal) allowed for evaluation of a
nonregional LN (submandibular)
0.5 cm in diameter. In
the other case, several nodes with gross metastases were
found. The lymphocyte composition of one of these in
volved nodes was determined. The gross tumor was dis
sected away, and the node was processed as described
under “
Materials and Methods.―No tumor cells could be
identified in the final lymphoid cell suspension.
A progression from NLN composition in the distant LN to
abnormal composition in the RLN was apparent. The small
LN from the same area as the RLN exhibited a cellular
profile intermediate between the 2 extremes. The large
n.onregional LN did not differ from normal, whereas the LN
grossly involved with tumor showed the lowest percentage
of T-cells and highest percentage of MIg-positive cells (Bcells). DLN from 5 other patients also demonstrated lym
phocyte subpopulations close to that in NLN (Table 1).
Radiated RLN contained a more normal proportion of
cells with the various markers than did nonmadiated RLN.
The radiated RLN from 2 patients had an average composi
tion of 61% T, 17% Fc, 15% C', and 12% MIg cells; while
radiated DLN consisted of an average of 63% T, 13% Fc,
15% C', and 10% MIg cells.
Table 2
Lymphoid
aprimary
subpopulations
in LN at various distances
from
C'receptor
cancer: percentage of T-ce!ls,Fc receptor cells,
cells, and Mig-bearing
LNT-cells cells in various
MIgPatient
larynxRLN
Fc
with squamous
25Small
(deep cervical)
17Distant
(deep cervical)
12Nonregional
(posterior cervical)
14lar)Patient(submandibu-
62
68
73
70
cancer of the
20
16
7
9
cancerRLN
with retromolar squamous
25Small
(submandibular)
55
23
19Distant
(submandibular)
66
13
12Tumor
(posterior cervical)
76
10
33ular)
involved(submandib- 46
21
1156
C'
19
13
10
11
24
15
12
18
NLN. The percentage of T-cells in normal human cervical
and axillany LN was significantly greaten than that found
among normal PBL (p < 0.01) (Table 1). This difference
cannot be accounted for by a higher percentage of macno
phages in the PBL (see below), for the proportion of T-cells
in PBL was not increased by removal of phagocytic cells on
polystyrene bead active adherence columns. The percent
age of Fc receptor cells in NLN was lower than that found in
normal PBL (p < 0.001) and, in contrast, in PBL Fc-positive
cells were less than MIg-beaning cells (p < 0.05). For deter
mination of whether this low number of Fc receptor cells
was due to blocking of Fc receptors in vivo by immunoglob
ulin, 2 experiments were undertaken. First, NLN cells were
washed with copious amounts of MEM HEPES for a mini
mum of 12 washes and surface markers (T, Fc, C', MIg)
showed no change.Second, NLN cellswere incubatedin
MEM HEPES without serum at 4°,23°,and 37°for periods up
to 24 hr to allow for the normal turnover of membrane
receptors and immunoglobulin-Fc receptor complexes. The
cells were also washed intermittently oven the 24-hr incuba
tion to wash out any immunoglobulin that might dissociate.
There was no increase in the percentage of Fc-positive
cells. Comparing NLN to PBL, the percentage of comple
ment receptor cells was unchanged (p > 0.05) while the
percentage of MIg-beaning cells was decreased (p < 0.05).
There was no significant difference among NLN from the
same person. Therefore between 2 and 7 LN were combined
to make up each sample. The number of gnanulocytes,
estenase-staining cells, and phagocytic cells was less than
1% in all NLN while the viability averaged 85.4 ±5.3%.
DISCUSSION
The NLN has long been known to consist of both T- and Bcell areas (24). The relative proportion of these cells in LN
can be estimated only crudely by histological on rosette
overlay techniques. Application of cell surface chanactemiza
tion techniques allowed us to define a consistent pattern of
alterations in lymphocyte subpopulations in RLN in patients
with squamous cancer of the head and neck.
LN immediately draining a localized cancer (RLN) showed
a clean distinction in lymphoid subpopulations as compared
to NLN. T-cell percentage was decreased, while B-cell per
centage (C' and MIg positive) was increased. This should
not be taken to mean that there was a decrease in absolute
T-ceII number but rather that themewas greater proliferation
or accumulation of these other cell types (Fc, C', MIg). In
animals with early implanted tumors, uniform increase in
RLN weight has been shown to occur (9). The increase in Fc
receptor cells (3.5-fold) in RLN could have been due to an
increase in T- on B-cell expression of this marker. When T
cells were purified, however, the number of Fc-positive cells
in this population was not increased. Surprisingly, all RLN
showed these changes, although differences in reaction
patterns as defined histologically are well described in
squamous cancer of the head and neck (14) as well as other
cancers (12, 13).
The differences between RLN and NLN cells should not
be taken as absolute differences, for this study was do
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RLN Populations in Cancer
signed to define NLN lymphoid subpopulations and to look
for evidence for changes from normal in RLN. Therefore,
the LN immediately adjacent to tumors were contrasted to
NLN. A continuum of LN composition exists as evidenced
by our studios on RLN and DLN from those same patients.
RLN will be exposed to tumor-related products prior to
vasculanization of minute tumors. Thus RLN may initiate
and modulate the initial host tumor relationship. The me
gional areas of expanded B-lymphocyte activity may play a
crucial moleas localized sites of antibody synthesis. While
antibody may arm certain Fc receptor cells to kill tumor
cells (20, 26), both NLN and RLN human lymphocytes, in
contrast to blood and spleen lymphocytes, are incapable of
mediating lysis of antibody-coated target cells (23, 28). On
the other hand, antibody complexed with tumor antigen has
been shown to be capable of blocking lysis of tumor cells by
cytolytically active lymphocytes (4, 15). In RLN high concen
trations of antibody and tumor antigen may well be focused
upon the RLN lymphocytes responsible for the development
of anti-tumor cell-mediated immunity leading to inhibition
of the early and effective expression of this activity.
Our data that normal cervical axillany LN were mainly
comprised of T-cells confirm the report of Holowiecki et al.
(16), who found 70 ±10% T-cells in normal abdominal LN.
The number of MIg-positive cells in NLN was less than in
peripheral blood on in the LN studied by Holowiecki et a!.
(22
± 16; p < 0.05).
Abdominal
LN may
be exposed
to
greater stimulation than can cervical or axillany LN due to
antigens entering through the gastrointestinal tract. Also,
their material came from patients with peptic ulcer disease
or cholelithiasis, and some of these nodes may have been
reactive, which would account for the wide mangein their
sample. Variations in immunofluorescont technique may
also be responsible for the difference. Recently, Samarut et
a!. (28) reported on the composition of 6 normal abdominal
lymph nodes. They also noted a T-ceII predominance (62 to
75%). B-cell (C' receptor lymphocytes and cells reactive
with an anti-B-cell antiserum) percentages were similar to
normal peripheral blood. Fc receptor lymphocytes were
markedly decreased in the NLN examined (0.6 to 4.3%) in
agreement with our findings.
Both NLN and RLN cell suspensions showed a consistent
and virtual absence of macrophages as defined by both
esterase stain and phagocytic function. Indeed, Zucher
Franklin (33) found 44% of PBL to be monocytes by bead
phagocytosis yet was unable to identify monocytes in the
LN cell suspensions studied. This lack of monocytes was
also noted by Holowiecki et a!. (16). Although LN trapping
and processing of antigen may be performed by lympho
cytes, the antigen-trapping cells are probably true macno
phages that could not be dissociated from the fibrous net
work of the LN on were present in very low numbers com
pared to lymphocytes.
ACKNOWLEDGMENTS
The authors would like to thank Dr. John L. Fahey, for his enthusiastic
support and helpful suggestions in preparing this manuscript, and Joanne
Bihr, for her excellent technical assistance. The anti-sheep RBC antisera
were a generous gift from Dr. Benjamin Bonavida.
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CANCER RESEARCH VOL. 37
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Lymphoid Subpopulation Changes in Regional Lymph Nodes in
Squamous Head and Neck Cancer
Andrew Saxon and John Portis
Cancer Res 1977;37:1154-1158.
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