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*
Liver Research Laboratories, Queen Elizabeth Hospital, Birmingham, United Kingdom; and
Department of Pathology, University of Birmingham Medical School, Birmingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To mount a successful immune response against a solid tumor, T cells
must first enter the tumor tissue and then recognize and respond to
tumor Ags. The mechanisms that regulate the recruitment of T cells to
tumors are poorly understood. In general, T cells must first recognize
and then adhere to endothelium before they can extravasate from the
circulation into tissue (8, 9, 10). This process is regulated by a
sequence of molecular interactions involving cell adhesion molecules on
both the T cell and the endothelium. The first step is a transient,
tethering interaction classically mediated by selectins that bind to
carbohydrate counterreceptors and induce the T cell to roll on the
endothelium (11). Other molecules can also mediate tethering, including
the 
4 integrins and their ligands VCAM-1 and mucosal
addressin cell-adhesion molecule-1 (MAdCAM-1) (12, 13). Strong,
secondary adhesion is mediated by lymphocyte integrins such as LFA-1
and VLA-4 and their respective endothelial ligands, ICAM-1, ICAM-2, and
VCAM-1 of the Ig superfamily. However, integrin-mediated adhesion
requires activation for efficient engagement of integrin ligands, and
it has been proposed that an additional step is required after
tethering in which T cells come into contact with activating factors at
the endothelial surface that trigger secondary, integrin-mediated
adhesion (14, 15). These factors can be either cytokines, particularly
those of the chemokine family, or cell surface molecules such as CD31
and CD73 (16). After secondary adhesion, the T cell extravasates across
the vessel wall in response to local chemotactic factors (14, 17, 18).
Thus, the combination of tethering (primary adhesion), triggering
(integrin activation signal), integrin-mediated adhesion (secondary
adhesion), and chemotactic factors are required to fulfill the
combinatorial requirements for T cell recruitment (14). The involvement
of particular combinations of molecules in the cascade can result in
the recruitment of selected subsets of T cells to specific tissues. For
example, the cutaneous lymphocyte Ag is expressed at high levels on T
cells that migrate to the skin, where it mediates binding to dermal
E-selectin (19) but not on T cells at other inflammatory sites such as
the liver (20). The integrin 4ß7, which is
expressed on low numbers of circulating T cells, is found on T cells
that migrate to the gut, where it binds to a receptor, MadCAM-1, that
is largely restricted to intestinal endothelium (21, 22). We have
recently shown that the endothelial adhesion molecule vascular adhesion
protein-1 (VAP-1), is constitutively expressed on liver vascular and
sinusoidal endothelium, where it supports T cell adhesion, thus
suggesting that it is involved in directing T cell recirculation to the
liver (23).
Although the precise mechanisms of T cell recruitment to tumors may differ from those involved in recruitment to inflamed tissue, it is likely that the same general principles apply. Thus, T cell recruitment to tumors will depend on the cellular adhesion molecules expressed by the T cell as well as the presence of appropriate endothelial ligands on tumor vessels. Once recruited to the tumor, the T cell must recognize and respond to tumor Ags (1). This process requires both recognition of tumor Ags presented by MHC molecules on the tumor cells and Ag-independent adhesive interactions that bring the T cell and tumor cells together and provide costimulation for T cell activation (24).
Our immunohistochemical studies indicate that hepatocellular carcinomas (HCC) are more heavily infiltrated by T cells than colorectal hepatic metastases (CHM), suggesting that these tumors are candidates for adoptive immunotherapy with TIL. Support for this hypothesis comes from murine models in which adoptive immunotherapy has been successful (25). Novel therapies for HCC are much needed because of the very poor prognosis of these tumors with conventional therapy (26). In the present study, we have confirmed that HCC are more heavily infiltrated by T cells than CHM and looked for differences in the expression of adhesion molecules on tumor endothelium that might account for these findings.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Samples of fresh liver tumor and macroscopically normal liver tissue from elsewhere in the same specimen were obtained from 25 patients (14 males, 11 females) with a median age of 61 yr (range 21 to 79) who underwent liver resection for primary or secondary liver tumors at the Queen Elizabeth Hospital, Birmingham, U.K. Ten patients had well-differentiated HCC, including one fibrolamellar variant. Three of the HCC patients had background cirrhosis secondary to chronic hepatitis B, primary biliary cirrhosis, and primary sclerosing cholangitis. Fifteen patients had CHM (four well-, nine moderately, and two poorly differentiated tumors). Samples of tumor and macroscopically normal liver tissue (taken at a distance of more than 5 cm from the tumor margin of the same specimen) were snap frozen in liquid nitrogen and stored at -70°C until used for immunohistochemical analysis and tissue-binding assays.
Monoclonal antibodies
The following mouse anti-human mAbs were used at saturating
concentrations for flow cytometry and immunohistochemistry: UCHT-1
(anti-CD3, IgG1; Dakopatts, Glostrup, Denmark); T4-4D7
(anti-CD4, IgG2a; Unipath, Bedford, U.K.); DK25 (anti-CD8,
IgG1; Dakopatts); 63D3 (anti-CD14, IgG1; a gift from Dr. S. Shaw,
National Cancer Institute, Bethesda, MD); EBM11 (anti-CD68, IgG1;
Dakopatts); 4KB128 (anti-CD22, IgG1; Dakopatts); ACT-1
(anti-CD25, IgG1; Dakopatts); UCHL-1 (anti-CD45RO, IgG2; a gift
from P. Beverly, University College, London, U.K.); MOC-1
(anti-CD56, IgG1; Dakopatts); L-78 (anti-CD69, IgG1; Becton
Dickinson, Mountain View, CA); Ber-T9 (anti-CD71, IgG1; Dakopatts);
F8/86 (anti-Factor VIII rAg, IgG1; Dako, Carpinteria, CA); CR3/43
(anti-HLA-Dr, IgG1; Dako); IELß7 ACT-1
(anti-4ß7 integrin; IgG1; a gift from
A. Lazarovits, University of Western Ontario, London, Canada); Ber-Act8
(anti-IELß7, IgG1; Dako); Leu-8
(anti-CD62-L, IgG1; Becton Dickinson); HP/1 (anti-CD49d, IgG1;
Coulter Immunology, Hialeah, FL); and 1.2B6 (anti-CD62E, IgG1; a
gift from D. Haskard Imperial College, London, U.K.). The following
mAbs were used at blocking concentrations of greater than 20 µg/ml of
purified Ab for assessing binding in in vitro tissue-binding assays:
1B2 (anti-VAP-1, mouse IgG1 isotype) was a gift from Dr. Marko
Salmi and Dr. Sirpa Jalkanen (Turku University, Turku, Finland); 84H10
(anti-CD54 (ICAM-1), mouse IgG1 isotype) was a gift from Dr. S.
Shaw; and 2G7 (anti-CD106 (VCAM-1), mouse IgG1 isotype) was
provided by Dr. W. Newman (Leukosite, Boston, MA). The following mAbs
were used at a concentration of 5 µg/ml per 106
cells to block T cell adhesion molecules: 25.3.1 (anti-CD11a, mouse
IgG1 isotype) used to block the -chain of LFA-1 was purchased from
Coulter, Luton, Bedford, U.K.; Kim249 (anti-CD11b, mouse IgG1
isotype) used to block Mac-1 was a gift from M. Robinson,
Celltech, Slough, U.K.; R.15/7 (anti-CD18, mouse IgG1 isotype) used
to block the ß-chain of LFA-1 was a gift from Dr. R. Rothlein
(Boehringer Ingelheim, Hartford, CT), CN; MAB13 (anti-CD29, mouse
IgG1 isotype) was kindly provided by Dr. K. Yamada (National Cancer
Institute, Bethesda, MD).
Immunohistochemistry
The phenotypic characterization of in situ TIL and the expression of endothelial adhesion molecules were studied by immunohistochemistry on 6-µm cryostat sections as described previously (20, 27). Tissue sections were fixed in acetone for 10 min at room temperature and then incubated with primary Ab, followed by secondary rabbit anti-mouse Ab, which was detected by an indirect alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, and the resulting enzyme complex developed with naphthol-AX and fast red. Sections were counterstained with Mayer’s haematoxylin. Incubations were done at room temperature for 45 min and sections were washed for 5 min with two changes of Tris buffer in between incubations. Normal tonsil sections were used as positive controls and sections stained with an irrelevant mouse primary mAb were used as negative controls. The enumeration of positive cells was conducted on 20 randomly selected high power fields (hpf) (magnification x400) using an ocular grid on every section. The mean ± SEM cells positive for each antigenic determinant was calculated for all patients in HCC and CHM. The intensity of endothelial staining on each section was graded from 0 to 3, where 0 = absent, 1 = weak, 2 = moderate, and 3 = strong staining. An overall mean score for the total number of patients in each group was then calculated for each endothelial adhesion molecule.
Lymphocyte isolation
TIL were isolated from fresh tumor tissues removed at surgery as described previously (28). Tumor tissues were immediately cut into small pieces, washed, and digested using RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 0.2% (w/v) collagenase type IV (Sigma, Poole, Dorset, U.K.) and 20% FCS (Life Technologies) for 2 to 3 h with continuous stirring at room temperature. The tumor digest was then passed through a nylon mesh to obtain a single cell suspension that was washed with PBS until the supernatant became clear. The single cell suspension was layered onto Ficoll-Hypaque (Lymphoprep; Nycomed, Oslo, Norway), centrifuged at 1600 rpm for 30 min at room temperature. TIL and tumor cells were recovered from the interface. Autologous PBL were isolated from heparinized venous blood obtained from the same patients immediately before surgery. PBL were separated by Ficoll-Hypaque centrifugation at 1600 rpm for 30 min at room temperature and then washed twice with PBS.
Culture of TIL and PBL
TIL and PBL were cultured in RPMI 1640 supplemented with 10% (v/v) FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin, 2 mM L-glutamine, and 1000 IU/ml human rIL-2 (Chiron, Harefield, Middlesex, U.K.) at 37°C in a humidified atmosphere with 5% CO2. Cultures were started in 24-well plates at 0.5 x 106 cells/ml and when the cell concentration exceeded 2 x 106/ml, cultures were transferred to T75 flasks for further expansion.
Isolation and culture of hepatic endothelial cells
Human intrahepatic endothelial cells were isolated from surplus liver tissue obtained from adult donor livers that had been reduced in size for transplantation into pediatric recipients as described previously (29, 30). Endothelial cells were isolated from approximately 150 g of human liver tissue after segmental perfusion with 5 mM calcium chloride, 0.05% collagenase H (Life Technologies), 0.025% dispase (Boehringer Mannheim, Mannheim, Germany), 0.0125% type 1-S hyaluronidase (Sigma), and 0.005% DNase (Boehringer Mannheim) in 10% HBSS. The cell suspension was filtered and centrifuged at 28 x g to pellet hepatocytes and the residual supernatant centrifuged at 717 x g to pellet nonparenchymal cells. The resulting pellet was washed and subjected to gradient centrifugation with 30% (w/v) metrizamide (Nycomed) before further separation using a JE-6B elutriator rotor (Beckman Instruments, High Wycombe, Buckinghamshire, U.K.) to isolate endothelial cells (31). The elutriated fraction was then resuspended in mouse anti-human CD31 (NIH31-1 used at 10 µg/ml) and positively immunoselected using 107 sheep anti-mouse IgG1 (Fc)-coated Dynabeads (Dynal U.K., Oslo, Norway). The immunoisolated cell fractions were resuspended in 2 ml of human endothelial basal growth medium, supplemented with penicillin and streptomycin (Life Technologies), 20% human AB+ serum (National Blood Transfusion Service, Edgbaston, Birmingham, U.K.), 10 ng/ml of vascular endothelial growth factor (VEGF), and 10 ng/ml hepatocyte growth factor (both from Bachem, Safron Walden, Essex, U.K.), plated onto collagen-coated six-well tissue culture plates (Costar, High Wycombe, Bucks, U.K.), and incubated at 37°C in a humidified atmosphere containing 3% CO2 in air. Once the cells had grown to subconfluence, they were characterized by immunocytochemistry and flow cytometry: >95% cells expressed the endothelial marker CD31 and >50% of cells expressed cytoplasmic VAP-1 (23). None expressed either the macrophage/leukocyte Ags (CD18 or CD45) or the fibroblast Ags as detected by mAb AS02 (Klein Fontenay 1; Dianova, Hamburg, Germany).
Flow cytometry
The phenotypic composition of freshly isolated and rIL-2-expanded TIL and autologous PBL were analyzed by two-color flow cytometry using standard techniques (32). Single cell suspensions (106 cells/ml) were incubated with 5 µl of primary unconjugated mouse mAb followed by a 1 in 20 dilution of FITC-conjugated F(ab')2 fragments of rabbit anti-mouse Ig (Dako). Thereafter, the cell suspension was incubated with normal mouse serum to saturate the free binding sites on the F(ab')2 fragments before a final incubation with phycoerythrin-conjugated anti-CD3 (Dako). All incubations were conducted at 4°C for 30 min and cells were washed twice with PBS (0.02% w/v sodium azide and 2% v/v FCS) in between incubations. The cell suspension was fixed with 1% paraformaldehyde and analyzed using the FACS 440 (Becton Dickinson). A lymphocyte gate was set to exclude dead cells and debris and at least 10,000 cells were analyzed in each sample. Mouse isotypes IgG1 and IgG2a were used as controls.
Tumor tissue-binding assay
We modified a previously published assay (23, 33, 34) to study the adhesion of rIL-2-expanded TIL and autologous PBL (cultured under the same conditions) to tumor endothelium on tissue sections. Cryostat sections (10 µm) of tumor tissue were cut onto poly-L-lysine-coated glass slides and fixed in acetone for 10 min before the study. TIL were preincubated with mouse anti-CD3 mAb for 30 min at 4°C and then washed twice with PBS and adjusted to a concentration of 107 cells/ml of medium (RPMI 1640 and 10% FCS). Sections were preincubated with control mAb or blocking mAb for 30 min at room temperature before addition of 150 µl of TIL suspension to each section with constant rotation (60 rpm) for 60 min at 4°C. Sections were gently washed with cold PBS to remove nonadherent lymphocytes and fixed in acetone for 10 min. Vascular endothelium and adherent lymphocytes were identified by anti-Factor VIII and anti-CD3 immunostaining, respectively, and developed with APAAP and fast red. The sections were counterstained with Mayer’s hematoxylin. The assays were done in the presence of a control mAb (S37) or blocking concentrations (>20 µg/ml) of the following mAb: 84H10 (anti-ICAM-1), 2G7 (anti-VCAM-1), 1B2 (anti-VAP-1), 25.3.1 (anti-CD11a), R15/7 (anti-CD18), and KIM 249 (anti-CD11b). These mAbs have all been shown to block adhesion in binding assays (23, 30, 32, 35, 36). Four tissue sections were used for each mAb per patient and the number of lymphocytes adherent to vascular endothelium (detected with anti-Factor VIII) was counted in both HCC and CHM using an ocular grid on every section. In addition, the number of lymphocytes adherent to sinusoidal endothelium and to vascular endothelium in the HCC sections were counted separately. Twenty high power fields (magnification x400) were randomly selected and counted on every tissue section. The number of lymphocytes adherent to endothelium in the presence of control mAb S37 defines 100% binding, and the number binding to vascular or sinusoidal endothelium in the presence of blocking mAb was expressed as a percentage of this.
To determine whether VAP-1-mediated adhesion of TIL to HCC endothelium is carbohydrate dependent, the tissue-binding assays were repeated in three cases after pretreatment of tissue sections with neuraminidase to remove sialic acid residues as described previously (37). Briefly, tissue sections were incubated with 5 mU of Vibrio cholerae-derived neuraminidase (Sigma) in 50 mM sodium acetate buffer solution (pH 5.5 with 100 mM NaCl and 5 mM CaCl) for 30 min in a humidified chamber at 37°C. After neuraminidase digestion, the sections were thoroughly washed with distilled water to remove remaining enzymes and subsequently used for tissue-binding assays as described above. Control sections were incubated with buffer solution only in the first step and subsequently with the appropriate mAb.
Adhesion to liver-derived endothelium in vitro
Human intrahepatic endothelial cells (between two and six
passages) were plated out and cultured to confluence in 48-well tissue
culture plates (for static assays) or 24-well plates for rotating
assays in medium containing 100 U/ml of TNF- for 24 h. The
static adhesion assays were performed according to an established
protocol as previously described (32). T cells were radiolabeled with
100 µCi of Na51CrO4, washed three times
in PBS, resuspended in RPMI 1640 containing 0.2% BSA, and added to the
confluent monolayers of endothelial cells at a final concentration of
2 x 106 cells/ml for 60 min at 37°C. At the end of
the assay, nonadherent cells were removed by washing with PBS and the
adherent cells lysed by incubation with 100 µl of 1% Igepal. Lysates
were collected and analyzed for 51Cr activity. A
modification of a previously described assay was used to determine
lymphocyte adhesion to endothelial cells under conditions of shear
stress (38). The assay was done as described for static adhesion except
that lymphocytes were added to endothelial cells that were cultured in
24-well plates under constant rotation at 60 rpm for 2 h at
37°C. Nonadherent cells were then gently aspirated and the wells
washed gently in PBS. Saturating concentrations of mAb to VAP-1 (1B2)
or a control mAb A7 were added to block adhesion.
Statistics
Results of positive cell enumeration are reported as mean ± SEM per hpf. Differences between groups were analyzed by the nonparametric Wilcoxon’s rank sum test. The level of significance was taken at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is a more intense T cell infiltrate in HCC when compared
with CHM (Figs. 1
and 2). There were
differences in the intensity and distribution of mononuclear cell
infiltrates in HCC and CHM. CD3+ cells were the predominant
infiltrating cell type in HCC, and focal aggregates were often seen in
close proximity to tumor vessels (Fig. 1A), whereas
comparable numbers of CD3+ T cells and macrophages were
seen in CHM (Fig. 2). The T cell
infiltrate was more intense in HCC, where there were significantly more
CD3+ T cells per hpf when compared with CHM (HCC mean
51 ± 10 positive cells per hpf compared with CHM 19 ± 4,
p < 0.01; Fig. 2). Both tumors contained a
predominance of CD4+ cells in the mononuclear cell
population (mean CD4/CD8 ratio 2.2 in HCC and 1.8 in CHM). In HCC,
CD8+ cells were found in the sinusoidal lumen and also in
contact with tumor cells (Fig. 1C). In CHM, few
CD3+ cells were detected in the parenchyma of tumor tissue
(Fig. 1B), but more intense T cell infiltrates were
observed at the tumor periphery (Fig. 1D). There were
few cells that were positive for the B cell marker CD22 in either tumor
type (Fig. 2).
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and 4)
Two-color flow cytometric analysis revealed that more than 90% of
freshly isolated TIL from both HCC and CHM were CD3+
and CD45RO+ (Fig. 3) and a
high proportion of TIL also expressed HLA-Dr and CD69, suggesting that
they are activated memory T cells (Fig. 3). However, only a small
percentage of the CD3+ cells in either TIL or PBL were
positive for the -chain of IL-2 receptor (CD25) or the transferrin
receptor (CD71) (Fig. 3). Very few freshly isolated TIL from either HCC
or CHM expressed L-selectin (CD62) compared with autologous PBL (Fig. 4A). Comparable numbers
of TIL and PBL expressed the 4ß7 integrin;
and up to 20% of CD3+ TIL in both tumors expressed
IELß7 (Fig. 4A). In
contrast, a higher proportion of CD3+ cells in both TIL and
PBL were positive for both the - and ß-chains of LFA-1 (CD11a and
CD18, respectively) and, to a lesser extent, VLA-4 (CD49a and CD29,
respectively) (Fig. 4B).
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The phenotypic compositions of cultured TIL and PBL from both
tumors used for in vitro adhesion assays are summarized in Table I. The phenotype of lymphocytes in
culture was monitored serially by two-color flow cytometry until they
reached phenotypic stability, which occurred after 4 wk of in vitro
culture. All the early TIL and PBL cultures were enriched in
CD4+ T cells, but three of seven HCC TIL became
CD8+ enriched in the later stages. The expression of
L-selectin was up-regulated in the early stages of culture but rapidly
declined after 2 wk and remained low in long-term culture.
CD3+ cells from both TIL and PBL cultures showed higher
expression of both the (92% ± 4)- and ß (90% ± 4)-chains of
VLA-4 in comparison to freshly isolated cells
(p < 0.01). TIL- and PBL-derived T cells used
for adhesion assays expressed high levels of CD11a, CD18, CD29, and
CD49a but low levels of L-selectin and CD11b according to flow
cytometric analysis (Table I).
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Immunohistochemical study of the expression of endothelial
adhesion molecules revealed differences between HCC, CHM, and adjacent
nontumoral liver (Table II). In the
autologous nontumoral tissues obtained from 15 patients without
background cirrhosis, there was strong expression of VAP-1 on
sinusoidal endothelial cells and moderate expression on hepatic
arteries, and portal and hepatic veins. In contrast, Kupffer cells,
hepatocytes, and biliary epithelial cells were all negative for VAP-1.
A similar staining pattern was observed for ICAM-1 on hepatic
endothelium, but in contrast to VAP-1, ICAM-1 was also strongly
expressed on Kupffer cells although the parenchymal cells were
negative. The pattern and intensity of both VAP-1 and ICAM-1 expression
were consistent in all the sections studied. There was diffuse but weak
expression of VCAM-1 on both the sinusoidal endothelium and Kupffer
cells and variable, focal VCAM-1 expression on the endothelium of both
hepatic and portal vessels. No E-selectin was detected on any structure
in any of the sections studied.
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). The exception was a fibrolamellar variant
of HCC, in which the sinusoidal space has been replaced by fibrous
tissue and immunoreactivity was detected only on the endothelium of
tumor vessels. The staining intensity of the sinusoidal expression for
VAP-1 was strong in 5 of 9 and moderate in 4 of 9 cases (Table II and
Fig. 5F). Similarly, the sinusoidal immunoreactivity
for ICAM-1 was strong in 5 of 9 and moderate in 4 of 9 cases (Table II
and Fig. 5E), although sections that were strongly
positive for ICAM-1 did not show corresponding VAP-1 staining
intensity. VAP-1 was detected on the endothelium of tumor vessels in
all 10 HCC cases; in 1 case this was strong, in 7 cases moderate, and
in 2 it was weak (Table II and Fig. 5C). ICAM-1
expression on tumor vessels in HCC was moderate in 3 of 10 and weak in
7 of 10 cases (Table II and Fig. 5B). In tumor
sections from 2 HCC patients, there were focal areas of poorly
differentiated tumor cells where the sinusoidal architecture was lost
and VAP-1 and ICAM-1 expression were observed only on the endothelium
of tumor vessels. In well-differentiated HCC, VAP-1 and ICAM-1 were
consistently expressed on sinusoidal lining cells and tumor
endothelium. Weak VCAM-1 expression was detected on the endothelium of
tumor vessels and sinusoids in 7 of 10 cases of HCC (Table II and Fig. 5, D and G). In contrast to the weak
endothelial expression, macrophages lying in the sinusoidal lumen with
appearance and distribution similar to Kupffer cells were strongly
positive for VCAM-1.
In contrast to HCC, the expression of endothelial adhesion molecules on
CHM was very different. VAP-1 was absent from tumor vessels in 14 of 15
cases of CHM, and in the other case only very weak, focal expression
was seen (Table II and Fig. 5J). ICAM-1 expression
was absent on tumor endothelium in 13 of 15 and only weakly positive in
the remaining 2 cases of CHM (Table II and Fig. 5I).
Tumor vessels in CHM showed weak focal VCAM-1 expression in 5 of 15 but
was absent in 10 of 15 cases (Table II and Fig. 5K).
E-selectin was not detected on endothelial cells from either tumor
type.
T cells cultured from TIL and PBL bind to tumor endothelium in HCC
via LFA-1/ICAM-1 and VAP-1 ligand/VAP-1 pathways (
Figs. 6–10)
The ability of T cells derived from TIL and autologous PBL
of HCC and CHM (cultured between 4 to 6 wk in 1000 IU/ml of rIL-2) to
bind tumor endothelium was examined by an in vitro tumor tissue-binding
assay conducted under rotary conditions (Fig. 6). Because sinusoidal endothelium and
vascular endothelium represent two distinct endothelial cell types in
HCC, we compared binding to vascular endothelium in HCC with binding to
vascular endothelium in CHM; binding to sinusoidal endothelium was
assessed separately (see below). HCC TIL-derived T cells bound readily
to tumor vascular endothelium on tumor sections (15 ± 4
cells/hpf; n = 7). Autologous PBL-derived T cells
cultured under the same conditions as TIL also bound to tumor
endothelium although in lower numbers (9 ± 3 cells/hpf;
n = 7) (Fig. 7A). When CHM TIL- and
PBL-derived T cells cultured under the same conditions were tested for
their ability to bind tumor vascular endothelium in autologous tumor
sections, very few cells bound (Figs. 6E and
7A). In addition, the number of cells bound to
vascular endothelium and sinusoidal endothelium in HCC sections was
enumerated separately (Fig. 6, A and B).
More cells bound to sinusoidal endothelium (TIL 34 ± 5, PBL
22 ± 5 cells/hpf) compared with vascular endothelium (TIL 15
± 4, PBL 9 ± 3 cells/hpf), and T cells derived from TIL bound
more readily to either endothelium than those from autologous PBL (Fig. 7B).
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and 8). Detailed analysis of binding between
sinusoidal and vascular endothelium revealed that similar adhesion
pathways were involved in binding to both structures. When used in
isolation, mAbs to ICAM-1 and VAP-1 had the greatest inhibitory effect
on binding to both types of endothelium (Fig. 6, G and
F, respectively); anti-VCAM-1 inhibited binding to a
lesser extent (Fig. 6, C and D).
Approximately 60% of TIL adhesion to tumor endothelium in HCC could be
inhibited by 84H10 (anti-ICAM-1 mAb) or 1B2 (anti-VAP-1 mAb)
alone compared with S37 control mAb, whereas 2G7 (anti-VCAM-1 mAb)
alone reduced adhesion by only 30% (Fig. 8). The combination of
blocking mAb against ICAM-1, VCAM-1, and VAP-1 reduced binding by 90%
(Figs. 6H and 8).
Because LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are both
ligands for ICAM-1, we investigated the relative contribution of these
molecules to T cell binding to HCC tumor endothelium. Flow cytometry
revealed that TIL-derived T cells are
CD11ahighCD11blowCD18high (Table I), and when tissue-binding experiments were repeated using blocking
concentrations of mAbs to CD11a, CD11b, or CD18 alone, TIL binding to
tumor endothelium was not inhibited by anti-CD11b mAb. In contrast,
anti-CD11a mAb reduced TIL binding by approximately 50% compared
with control, and anti-CD18 mAb inhibited binding to a similar
extent as anti-ICAM-1 mAb (Fig. 9).
These results suggest that LFA-1 on TIL is the major ligand for ICAM-1
on HCC tumor endothelium.
VAP-1 in normal tissue is heavily sialylated and the sialic
acid residues are required for lymphocyte adhesion to VAP-1 in lymph
node high endothelial venules (HEV) (37). To determine whether
lymphocyte binding to VAP-1 on tumor endothelium is also dependent on
sialic acid, we repeated the tissue-binding studies after treating
the tumor tissue with neuraminidase. Adhesion was reduced by the
presence of 1B2 alone when compared with the control mAb S37 (control,
19.3 ± 1.9; 1B2, 10.5 ± 9.4 cells/hpf), and neuraminidase
alone also reduced adhesion (4.5 ± 0.7 cells/hpf), but
the combination of 1B2 and neuraminidase did not reduce adhesion
further (3.9 ± 0.6 cells/hpf) (Fig. 10).
TIL bind to VAP-1 on human hepatic endothelial cells in
culture under shear stress (Fig. 11)
To determine whether VAP-1 supports shear-dependent adhesion, binding studies were conducted using monolayers of endothelial cells isolated from human liver that expressed VAP-1 on the cell surface (data not shown). mAb 1B2 (anti-VAP-1) failed to inhibit adhesion to human hepatic endothelial cells under static conditions but reduced adhesion by up to 40% when assays were conducted under constant rotation. In contradistinction, 1B2 had no effect on either static or shear-dependent adhesion of the same TIL to the dermal microvascular cell line HMEC-1, which does not express VAP-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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If TIL are to mediate antitumor responses, they must recognize and bind to tumor endothelium before migrating from the circulation into the tumor itself (42). Little is known about the expression of endothelial adhesion molecules in either primary or secondary liver tumors. Our results demonstrate that the phenotype of tumor endothelium in HCC differs markedly from that in metastatic colorectal carcinoma and that these differences are probably crucial in regulating T cell recruitment. With regard to adhesion molecules, endothelium in HCC is phenotypically and functionally similar to that in normal liver distant from the tumor. HCC contains two distinct types of endothelium; sinusoidal-like endothelial channels that run between the tumor cells and vascular endothelium on tumor vessels. Both of these endothelia stained strongly for ICAM-1 and VAP-1, two endothelial adhesion molecules that are found on sinusoidal and vascular endothelium in nonneoplastic liver (23, 43, 44, 45). In contradistinction, endothelium in the CHM did not express VAP-1 and only expressed ICAM-1 weakly. We have previously demonstrated that VAP-1 is expressed constitutively in the liver where it can support binding of T cells to hepatic endothelium via an as yet unknown T cell ligand (23). The results of the present study, in which VAP-1 supported TIL adhesion to tumor endothelium in HCC, suggest that lymphocyte adhesion to endothelium in primary liver tumors and normal liver may be regulated by similar mechanisms.
VAP-1 is a human endothelial adhesion molecule (34, 37), originally described by Salmi et al. (36) and Salmi and Jalkanen (37), which supports shear-dependent lymphocyte binding to HEV in lymph nodes. We observed that mAb to VAP-1 inhibited T cell adhesion both to the sinusoidal-type endothelium and to tumor vessels in tissue sections of HCC. Adhesion could also be blocked using anti-ICAM-1 mAb and, to a lesser extent, anti-VCAM-1 mAb. Recent studies have proposed that VAP-1 mediates initial lymphocyte interactions with endothelium, suggesting that VAP-1 and ICAM-1 could play complementary roles in lymphocyte recruitment to HCC by mediating adhesion at different stages of the adhesion cascade (23, 46, 47). To pursue this, we tested TIL adhesion to monolayers of human liver-derived endothelial cells that express low but detectable levels of VAP-1 on their cell surface (29). VAP-1 supported adhesion to these endothelial cells only under conditions of shear induced by rotation; it had no effect in standard static adhesion assays. These observations are consistent with the recent report that VAP-1 mediates initial, primary interactions between lymphocytes and mesenteric venules (36). It has been suggested that lymphocytes can bypass the need for a tethering requirement in low flow systems such as the hepatic sinusoids. However, recent intravital studies suggest that lymphocytes interact with hepatic sinusoidal endothelium in a two-stage process (48), and we think it is likely that similar mechanisms will apply to the sinusoids in HCC. The failure of sinusoidal endothelium, both in normal liver and hepatoma, to express selectins (20, 48) suggests that VAP-1 might play a particularly important role in mediating primary adhesion to these specialized vessels. Thus, VAP-1 on HCC endothelium could bring the circulating lymphocyte into contact with the vessel wall, allowing subsequent secondary adhesion to be mediated via LFA-1 (which is expressed at high levels on the TIL in HCC) and ICAM-1, which is abundant on tumor endothelium. The weaker expression of VCAM-1 on tumor endothelium and the less marked inhibition of lymphocyte binding with mAb against VCAM-1 when compared with anti-ICAM-1 suggest that VCAM-1 is less important, although in vivo studies are required to confirm this.
The lymphocyte receptor for VAP-1 is not known but previous studies have suggested that it acts independently of L-selectin and recognizes sialic acid residues on VAP-1 in tonsillar and lymph node HEV (36, 37). TIL binding to VAP-1 in HCC is also L-selectin independent because very little L-selectin is expressed by the TIL used for adhesion assays in this study. Furthermore, treatment of hepatoma tissue sections with neuraminidase abolished VAP-1-dependent adhesion, suggesting that adhesion to VAP-1 in hepatoma is also dependent on the presence of sialic acid residues (36, 37).
In contrast to HCC, we were unable to demonstrate any appreciable TIL binding to tumor vessels in the hepatic metastases from colorectal carcinomas, a finding that is consistent with the very low levels of adhesion molecules expressed by vessels in these tumors. The inability of endothelium in CHM to support T cell adhesion may be responsible for the paucity of infiltrating T cells in this tumor. It is possible that tumor-derived factors actively suppress endothelial activation in CHM, thereby protecting the tumor from immune attack as has been proposed for other tumors (49).
The expression of VAP-1 on HCC tumor endothelium is of great interest. VAP-1 is constitutively expressed on hepatic endothelium (23, 34), one of the few sites where VAP-1 expression is detected in the absence of inflammation. Although VAP-1 expression is up-regulated at inflammatory sites, including the gut and skin (50, 51), VAP-1 expression is very low in noninflamed mucosal vessels (50). In the present study, we detected strong expression of VAP-1 on the sinusoidal endothelium of nontumor autologous liver and also on the tumor vessels and sinusoids within the hepatomas. VAP-1 expression was absent in the colorectal metastases. This previously unreported finding provides further evidence that VAP-1 expression is characteristic of liver-derived endothelium. The fact that VAP-1 was expressed on endothelium in primary, liver-derived tumors but not in secondary tumors within the liver suggests that the anatomical position of the tumor within the liver is not sufficient in itself to induce VAP-1 expression. It is likely that the liver-cell origin of HCC is a crucial factor in determining the phenotype of the tumor endothelium. One factor that may be important in regulating the differentiation of hepatoma endothelium is VEGF, which we have recently shown is required to maintain VAP-1-expressing, human hepatic sinusoidal endothelial cells in culture (29). Other groups have reported that hepatoma cells express VEGF at the gene and protein levels (52), and our own unpublished observations show much stronger expression of VEGF in primary compared with secondary liver tumors (K. F. Yoong and D. H. Adams, unpublished observations). Thus, HCC-derived VEGF could be responsible for the distinctive phenotype of HCC tumor endothelium. The alternative explanation that endothelial activation in HCC is a consequence of cytokines derived from the large numbers of infiltrating T cells seems unlikely because tumor endothelium showed strong expression of VAP-1 and ICAM-1 even in areas that were not heavily infiltrated by T cells.
E-selectin was uniformly absent from both nontumoral and tumor tissue in both tumor types. Recent studies suggest that selectins are not involved in leukocyte interactions with inflamed hepatic endothelium (20, 48). However, E-selectin is expressed on hepatic vascular endothelium at sites of acute and chronic inflammation (20), and its absence from tumor endothelium might suggest active suppression by tumor-derived factors, as has been described for VCAM-1 and melanoma (49). TGF-ß could be one such factor because several human tumors produce it, and it can inhibit the expression of E-selectin on human endothelial cell lines in vitro (53).
We also looked for potential homing receptors on the TIL. T cells in
both tumors were CD62Llow and CD45ROhigh,
suggesting that they are memory cells. Two ß7 integrins
have been implicated in the recruitment and retention of T cells at
mucosal sites within the gut and might therefore be involved in the
recruitment of T cells to liver tumors. The
4ß7 integrin mediates the binding of
gut-tropic memory T cells to MadCAM-1, which is selectively expressed
on mucosal endothelium in the intestines (21, 54). The
IELß7 integrin is expressed on
intraepithelial T cells and mediates binding to E-cadherin on
intestinal epithelial cells, thereby retaining T cells in the
epithelium (55). However, ß7 integrins were detected on a
small percentage of TIL in either HCC or colorectal metastases. It thus
seems unlikely that they play a major role in T cell recruitment to
liver tumors. In the light of recent observations that
IELß7 is expressed on T cells within
primary colorectal tumors, the lack of
IELß7 expression of TIL in CHM is
surprising and may be related to down-regulation of E-cadherin on
metastatic colorectal tumors (56, 57).
In summary, the results of this study show that HCCs are more heavily infiltrated with T cells than hepatic metastases from colorectal carcinoma. This is possibly a consequence of the greater expression of functional adhesion molecules on endothelium in HCC, which is phenotypically similar to activated, nontumoral hepatic endothelium. In contrast, tumor endothelium in the hepatic metastases expressed low levels of adhesion molecules and failed to support T cell adhesion. The strong expression of VAP-1 on tumor endothelium in HCC supports our previous hypothesis that VAP-1 is an important hepatic endothelial adhesion molecule. We propose that TIL are recruited to hepatomas via interactions with VAP-1 (which mediates primary, tethering interactions) and ICAM-1 (mediating firm adhesion) on tumor endothelium. Why these T cells fail to suppress tumor growth is not known but will be important for the development of immunotherapy strategies for HCC. However, a better understanding of the mechanisms of T cell recruitment to tumor tissue will facilitate the generation of antitumor effector cells with the appropriate adhesion molecules to allow them to home to the tumor in adoptive immunotherapy. Such strategies will help to overcome the need for infusion of such large numbers of lymphocytes as is currently required to ensure that sufficient numbers of lymphocytes reach tumor deposits in melanoma (58).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Prof. D. H. Adams, Liver Research Laboratories, Clinical Research Block, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, U.K.
3 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocytes; HCC, hepatocellular carcinoma; CHM, colorectal hepatic metastasis; VAP-1, vascular adhesion protein-1; VEGF, vascular endothelial growth factor; MAdCAM, mucosal addressin cell-adhesion molecule-1; VLA, very late antigen; APAAP, alkaline phosphatase antialkaline phosphatase; hpf, high power field; HEV, high endothelial venule.
Received for publication February 10, 1997. Accepted for publication December 23, 1997.
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4ß7 on a subset of human CD4+ memory T cells with hallmarks of gut-trophism. J. Immunol. 151:717.[Abstract]
