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*
Education and Research Centre and
Liver Unit, St. Vincent’s Hospital, Dublin, Ireland; and
Department of Medicine and Therapeutics, University College, Dublin, Ireland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, IL-2, and/or IL-4, but
little or no IL-5, while NK cells produced IFN- and/or TNF- only.
The majority of NT cells produced inflammatory (Th1) cytokines only;
however, 
6% of all hepatic T cells, which included 5% of V24
TCR-bearing NT cells and 2% of 
TCR+ cells,
simultaneously produced IFN- and IL-4. The existence of such large
numbers of cytotoxic lymphocytes with multiple effector functions
suggests that the liver is an important site of innate immune
responses, early regulation of adaptive immunity, and possibly
peripheral deletion of autologous cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3-fold lower surface density of the ß TCR and the
NKR-P1 receptor NK1.1 (3, 4). NK1.1+
T cells are characterized by their potent effector function, as
evidenced by their ability to lyse various tumor cells in the absence
of prior antigenic stimulation, and to rapidly produce high levels of
Th1 and Th2 cytokines, including IFN- and IL-4, upon activation
through their TCR or NK1.1 molecules (5, 6, 7). A feature of
these cells is that they frequently express a highly restricted TCR
repertoire consisting of an invariant V14J281 -chain in
association with a limited number of Vß-chains (8),
which, unlike conventional MHC-restricted T cells, are positively
selected by and recognize nonpeptide Ags in the context of CD1
(9, 10).
Studies in humans have also identified lymphocyte populations that
coexpress ß or TCRs and various NK receptors, including
CD16, CD56, CD69, CD161 (NKR-P1A), and/or killer cell inhibitory
receptors (KIR)6 for
MHC class I (11, 12, 13, 14). These include a human NKT cell
population that coexpresses CD161 and a TCR that is structurally
homologous to the murine V14J281 TCR chain, V24-JQ
(8), which, like their murine V14J281 T cell
counterparts, can recognize nonpeptide Ags presented by CD1d
(15, 16, 17) and can be induced to produce both IFN- and
IL-4 (15, 18). T cells expressing NK cell receptors are
particularly abundant in the normal human liver (2), and
we have recently demonstrated that a significant proportion of hepatic,
but not peripheral, CD3+ cells can be induced to
lyse NK-sensitive target cells (19). In these experiments,
the effectors resided in a
CD3+CD56+ cell population
that makes up approximately one-third of all hepatic
CD3+ cells, but only 2% of peripheral blood T
cells. Human hepatic CD56+ T cells are a
heterogeneous population of lymphocytes that include
CD4+, CD8+ and
double-negative CD4-CD8-
cells expressing either ß or TCRs and Ag-experienced CD45RO
and CD56dim phenotypes. They include
CD3+CD161+ NKT cells, some
of which have invariant V24-JQ TCR chains, as well as
CD161-negative T cells expressing other NK receptors. Since the
expression of CD56 serves better than CD161-positivity to define an NK
receptor-positive T cell subpopulation that is characteristic of the
liver (19), we have defined "hepatic natural T (NT)
cells" as CD3+CD56+
cells, rather than
CD3+CD161+ cells.
The above observations suggest that the human liver is a major site of NT cell activity and may thus be a uniquely specialized organ of the immune system. However, the functions of hepatic NT cells are unknown. By analogy with murine NKT cells, they are likely to have innate (20), antitumor (21, 22), and regulatory (6, 7) roles, with multiple activities that are under the control of the accessory cells and soluble factors present in their local microenvironment (23, 24, 25, 26, 27, 28). Therefore, we have investigated the effector functions of human hepatic NT cells immediately after isolation from normal adult human liver specimens. By comparing the cytotoxic functions and cytokine secretion profiles of hepatic CD3+CD56+ NT cells with those of hepatic NK cells (CD3-CD56+) and conventional hepatic T cells (CD3+CD56-), we show that the NT cell fraction is capable of potent TCR-mediated and NK-like cytotoxicity, and that these cells rapidly produce proinflammatory, Th1 and Th2 cytokines. We further show that multiple subpopulations of hepatic NT cells are capable of simultaneously producing Th1 and Th2 cytokines. These results suggest that the adult liver is an important site of innate immune recognition and effector function as well as the early regulation of adaptive immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal liver tissue (100–300 mg biopsy specimens) was obtained from 18 adult donors at the time of liver transplantation. Liver biochemistry and histology were normal in all cases. Single hepatic cell suspensions that were enriched for mononuclear cells (MNC) were prepared using a method based on mechanical dissociation and collagenase treatment, as described previously (19). PBMC were prepared from 10 healthy adult donors by Lymphoprep density gradient centrifugation (Nycomed, Oslo, Norway). Ethical approval for this study was obtained from the Ethics Committee at St. Vincent’s Hospital (Dublin, Ireland).
Abs and flow cytometry
Anti-CD3 FITC, anti-CD3 peridin chlorophyll protein (PerCP),
anti-CD56 PE, anti-IFN- FITC, anti-TNF- FITC,
anti-IL-2 FITC, anti-IL-4 PE, anti- TCR biotin,
isotype-matched anti-IgG controls (FITC, PE, and PerCP) and
streptavidin PerCP were obtained from Becton Dickinson (Oxford, U.K.).
Unconjugated anti-CD3 (HIT3a), anti-CD56 biotin, anti-IL-4
FITC, and anti-IL-5 PE were obtained from PharMingen (Oxford,
U.K.). Anti-V24 TCR biotin was obtained from Coulter-Immunotech
(Marseille, France). Phenotypic analysis of hepatic lymphocyte (HL)
subsets (Fig. 1
, A and
B) was performed by three-color mAb staining and flow
cytometry using a FACScan (Becton Dickinson) and analysis using
CellQuest software (Becton Dickinson).
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Human recombinant IL-2, BSA, sodium azide, PMA, ionomycin, PHA, saponin, and brefeldin A were obtained from Sigma (Poole, U.K.).
In vitro stimulation and culture of HL
MNC-enriched hepatic cell preparations (0.1–1 x
106 viable cells) were plated out in 96-well
round-bottom or 24-well flat-bottom plates (Nunc, Kamstrup, Denmark)
and cultured with equal numbers of irradiated (3000 rads
irradiation) allogeneic PBMC and 5 µg/ml PHA in complete RPMI medium
(RPMI 1640 containing 25 mM HEPES, 2 mM L-glutamine, 50
µg/ml streptomycin, 50 U/ml penicillin, and 10% locally produced
heat-inactivated human serum) (29). Human recombinant IL-2
(30 U/ml) was added on day 3, and the cells were harvested for
functional analyses on day 5. Flow cytometric analysis of 5-day
cultures revealed negligible expansion or phenotypic changes to the
cells. Expansion of HL was achieved by restimulation as above every
7–10 days with the addition of IL-2 on days 3, 5, and 7
(29). Long-term culture of these cells resulted in the
selective expansion of CD3+ cells.
Isolation of HL subpopulations
Freshly isolated, in vitro-stimulated, and in vitro-stimulated
and expanded hepatic NK cells, T cells, and NT cells were tested as
effectors in cytotoxicity assays. Cells were separated into
CD3-CD56+ (NK cells),
CD3+CD56- (T cells), and
CD3+CD56+ (NT cells)
fractions using mAb-coated magnetic beads (Macs Magnetic Microbeads,
Miltenyi Biotec, Bergisch Gladbach, Germany). Two approaches were
employed. Double sorting of cells according to their expression of CD3
and CD56 was performed by labeling the cells with an FITC-conjugated
mAb against CD3, followed by the magnetic separation of the labeled
CD3+ cells from the unlabeled
CD3- cells using anti-FITC microbeads. The
magnetic beads were then removed using the MultiSort Kit (Miltenyi
Biotec). The CD56+ and
CD56- cells were subsequently separated from
these preparations using anti-CD56 microbeads.
CD3+CD56+ cells were also
purified by first selectively expanding the CD3+
cells in vitro, as described above, and then separating them into
CD56+ and CD56-
populations using anti-CD56 microbeads. The purity of isolated
fractions was assessed by flow cytometry (Fig. 1
, C–E), and
only preparations whose purities were >95% were used for functional
studies.
Cytotoxicity assays
NK and T cell cytotoxic function was assessed in 4-h
51Cr-release assays (29) using
fresh, in vitro-stimulated, or stimulated and expanded effector cell
populations, which were isolated by immunomagnetic bead separation, and
added to the target cells at E:T ratios of 1:1 to 100:1. In NK
cytotoxicity assays, the cytolytic activities of freshly prepared,
freshly isolated HL subsets against the target cell line, K562, were
measured. For the measurement of lymphocyte-activated killing (LAK)
activity, HL were first stimulated in vitro and cultured with IL-2 for
5 days before using as effectors in cytotoxicity assays against K562
cells. The FcR+ murine mastocytoma cell line
was used as the target cell for mAb-redirected cytotoxic killing using
cultured HL subsets as effectors with cross-linking mAbs to the T cell
or NK cell receptors CD3 or CD56 (12). In vitro expansion
of the effectors was required to obtain sufficient numbers of T cells
and NT cells for use in redirected cytotoxicity assays, but since
CD3-negative cells were not expanded by our method, NK cells were taken
from HL cultured for 5 days only. mAbs were incubated with the effector
cells for 30 min at 4°C and washed twice before adding the target
cells. Percent specific lysis was expressed as: [(cpm of sample
- cpm of spontaneous release)]/[(cpm of maximum release - cpm
of spontaneous release)].
Stimulation of cells and staining for intracellular cytokines
Freshly isolated MNC-enriched hepatic or peripheral blood cells
were suspended in complete RPMI medium at a density of 0.5–1 x
106 cells/ml and stimulated for 6 h in
24-well plates (Nunc; 0.5 ml/well) at 37°C in 5%
CO2. Cells were stimulated with either 10 ng/ml
PMA plus 1 µg/ml ionomycin or with plate-bound anti-CD3 mAb (10
µg/ml HIT3a bound to plates by incubation for 6 h at 37°C in
0.1 M Na2HCO3) in the
presence of 1 ng/ml PMA. As controls, unstimulated cells were treated
similarly. Brefeldin A (10 µg/ml), an inhibitor of protein
translocation from the endoplasmic reticulum to the Golgi apparatus,
was added to the cells for the last 4 h. After stimulation or
incubation without stimulators, cells were washed twice with PBS
containing 0.07% BSA and 0.02% sodium azide and stained for 15 min at
room temperature with mAbs against cell surface CD3, CD56, V24 TCR,
TCR, or isotype-matched controls. For the detection of TNF-,
IFN-, IL-2, and IL-4 (FITC-conjugated mAbs) production by HL
subsets, anti-CD3 PerCP and anti-CD56 PE were used for
cell-surface staining. For the detection of IL-5 (PE-conjugated), the
cell surface was stained with anti-CD3 FITC and anti-CD56
biotin. For the simultaneous detection of IFN- (FITC-conjugated mAb)
and IL-4 or IL-5 (PE-conjugated mAbs), cell surface staining was
performed using anti-CD3 PerCP, anti-CD56 biotin,
anti-V24 TCR biotin, or anti-TCR biotin. Cells were
then washed twice with PBS-BSA-azide buffer and stained with detection
reagents (streptavidin PerCP) where required before staining for
intracellular cytokines.
For the intracytoplasmic staining of cytokines, cells were fixed with
0.5 ml 4% paraformaldehyde at room temperature for 10 min, washed with
PBS-BSA-azide buffer, and permeabilized with 1 ml 0.2% saponin in
PBS-BSA-azide buffer for 10 min at room temperature. Cells were
pelleted and incubated with anti-cytokine mAbs (0.1 µg
anti-TNF- FITC, anti-IFN- FITC, anti-IL-2 FITC,
anti-IL-4 FITC, anti-IL-4 PE, or anti-IL-5 PE in 50 µl
0.2% saponin) for 30–60 min. Finally, the cells were washed with
PBS-BSA-azide buffer and resuspended in PBS-BSA-azide buffer for
immediate analysis or in 1% paraformaldehyde for overnight storage
before analysis. Coexpression of cell surface molecules and
intracytoplasmic cytokines was detected by three-color flow cytometry
(FACScan, Becton Dickinson) and analyzed using CellQuest (Becton
Dickinson) software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of cells from 100 to 300 mg liver specimens yielded
1–2.5 x 106 MNC. Flow cytometric analysis
of CD3 and CD56 expression was used to define conventional T
cells (CD3+CD56-),
NK cells (CD3-CD56+), and
NT cells (CD3+CD56+) (Fig. 1, A and B). Analysis of freshly isolated hepatic
MNC revealed that NK cells and NT cells were significantly more
frequent among HL compared with PBL (Fig. 1, A and
B), as we have previously reported (2, 19).
Flow cytometry of long-term HL cultures revealed that the in vitro
expansion of these cells resulted in the selective expansion of
CD3+KIR- cells; therefore,
functional assays using cultured NK cells were performed using cells
that were cultured for 5 days only, conditions which resulted in
negligible expansion or phenotypic changes to the cells.
Hepatic NT cells are capable of LAK, but not NK, cytotoxic activity
Freshly isolated or stimulated HL were separated into
CD3-CD56+ (NK cells),
CD3+CD56- (T cells), and
CD3+CD56+ (NT cells)
fractions using immunomagnetic beads (Fig. 1, C–E), and
each fraction was tested for its ability to lyse K562 cells in chromium
release assays. When freshly isolated cells were used as effectors,
only the NK cell fraction demonstrated lytic activity against this
target cell line, whereas NT cells and T cells did not (Fig. 2A). However, after
stimulation in the presence of IL-2, both the NK and NT cell fractions,
but not conventional T cells, exhibited cytotoxicity against K562
targets (Fig. 2B), indicating that the hepatic NT cell
fraction contains precursors of LAK cells.
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Cultured HL were separated, using magnetic beads, into NK cells, T
cells, and NT cells (Fig. 1, C–E) and tested for their
ability to lyse murine FcR+ P815 cells in
redirected cytotoxicity assays in the absence or presence of
cross-linking mAbs to human CD3. Fig. 2C shows that purified
hepatic CD3-CD56+ cells
(NK cells) were capable of lysing P815 cells whether or not any Abs
were present. This intrinsic cytotoxic activity was found to be the
result of in vitro stimulation of NK cells (LAK activity) since little
or no cytotoxicity was observed when freshly isolated
CD3-CD56+ cells were used
as effectors against P815 cells in the absence of cross-linking mAbs
(data not shown). Hepatic T cells
(CD3+CD56-) and NT cells
(CD3+CD56+) did not possess
LAK activity against P815 targets (Fig. 2C), but when
cultured HL fractions were used as effectors in redirected cytotoxicity
assays, CD3 cross-linking led to cytolysis of P815 targets, indicating
that both hepatic NT cells and conventional T cells are capable of
TCR-mediated cytolysis (Fig. 2D). No cytolysis of P815 cells
was observed after cross-linking with anti-CD56 or isotype-matched
control mAbs (data not shown). Since in vitro expansion of HL was
required to obtain sufficient numbers of T cells and NT cells for use
as effectors in these assays, we have not determined whether freshly
isolated HL subsets could also lyse P815 cells in redirected assays.
These results indicate that, like conventional T cells, cultured
hepatic NT cells are capable of TCR-mediated cytolysis, but, in
contrast to T cells, these cells can also be activated through
receptors that mediate LAK activity.
Cytokine production by hepatic and peripheral T cells, NT cells, and NK cells
Freshly isolated hepatic MNC were stimulated for 6 h with PMA
and ionomycin or plate-bound anti-CD3 mAb, or incubated in the
absence of stimulator, and the frequencies of cytokine-producing HL
subsets were determined by flow cytometry after mAb staining for
surface CD3 and CD56 and intracellular IFN-, TNF-, IL-2, IL-4,
and IL-5. Following stimulation with PMA and ionomycin, cytokine
production by NK cells, T cells, and NT cells was determined by
three-color flow cytometry of gated
CD3-CD56+,
CD3+CD56-, and
CD3+CD56+ cells,
respectively, as shown in Fig. 3. The
numbers of cells expressing CD3 and CD56 were not changed by these 6-h
stimulations. Stimulation of the cells by CD3 cross-linking resulted in
the blocking of CD3, thus interfering with the subsequent detection of
this molecule by flow cytometry (Fig. 4).
Since only the cells that express surface CD3 would produce cytokines
as a result of activation by anti-CD3 mAb, the frequencies of NT
cells that produced cytokines were calculated as the percentages of HL
that expressed CD56 and produced cytokines upon stimulation through
CD3, divided by the percentages of unstimulated HL that expressed the
CD3+CD56+ phenotype (Fig. 4). Similarly, the frequencies of cytokine-producing T cells were
calculated as the percentages of HL that were
CD56- and produced cytokines upon CD3
cross-linking divided by the percentages of unstimulated HL expressing
CD3+CD56- phenotypes (Fig. 4). In all experiments, background cytokine production by similarly
gated unstimulated cells or cells incubated with 1 ng/ml PMA was
negligible, but values obtained were nevertheless subtracted from those
obtained with stimulated cells to avoid subjectivity when defining
positivity and negativity for cytokine production (Figs. 4 and 5).
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, IFN-, IL-2,
IL-4, and IL-5 are summarized in Fig. 5, and the mean percentages
positivity for these cytokines are compared with those obtained with
peripheral blood NK and T cells in Table I. Significant and comparable proportions
of hepatic NT cells and T cells were capable of producing TNF-,
IFN-, IL-2, and IL-4, but not IL-5, upon PMA and ionomycin
stimulation or CD3 cross-linking (Fig. 5). The percentages of cells
expressing these cytokines were higher after stimulation with PMA and
ionomycin than after CD3 cross-linking. In all donors examined, the
majority (57–92%) of hepatic NT cells produced IFN-, while smaller
proportions produced TNF- (38–84%), IL-2 (32–61%), IL-4
(1–15%), and IL-5 (0–2%) (Fig. 5). Cytokine production by PMA and
ionomycin-stimulated hepatic NK cells was limited to TNF- (8.7%)
and IFN- (53%) (Fig. 5). The proportions of hepatic NK cells and T
cells that produced TNF-, IFN-, IL-2, IL-4, and IL-5 upon PMA +
ionomycin or anti-CD3 mAb stimulation were comparable to those of
peripheral blood NK and T cells (Table I).
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To examine coexpression of Th1 and Th2 cytokines by individual HL
subsets, fresh MNC-enriched hepatic cells were stimulated with PMA and
ionomycin and stained for surface expression of CD3, CD56, V24 TCR,
or TCR and intracytoplasmic production of IFN- and IL-4 or
IL-5. Fig. 6A shows that the
proportions of hepatic CD3+,
CD56+, V24 TCR+, or
TCR+ cells that produce either or both of
these cytokines could be determined using three-color flow cytometry
with gating on the cells expressing the surface markers. The
proportions of these HL subsets from five normal liver specimens that
were capable of simultaneously producing IFN- and IL-4 or IFN-
and IL-5 are shown in Fig. 6B. While the numbers of cells
producing IL-5 were very low, up to 12% (mean 6%) of
CD3+ HL simultaneously produced IFN- and IL-4.
This Th0 cell population included up to 11% (mean 5.1%) of V24
TCR+ HL, which accounted for a mean of 2.7% of
the total hepatic CD3+ T cell compartment. The
mean proportions of CD56+ cells and
TCR+ cells that demonstrated Th0 cytokine
profiles were lower (means 1.0% and 1.6%, respectively). The majority
of IL-4-producing cells in the CD3+ (82.1%),
CD56+ (56.8%), V24+
(87.0%), and TCR+ (59.8%) HL subsets also
produced IFN-, indicating that most hepatic IL-4-secreting cells
exhibit Th0 phenotypes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of the present study also indicate that the human liver
contains lymphocytes capable of the rapid secretion of proinflammatory,
inflammatory (Th1 or Tc1), and helper (Th2 or Tc2) cytokines.
Significant numbers of hepatic
CD3+CD56- T cells and
CD3+CD56+ NT cells can
produce TNF-, IFN-, IL-2, and/or IL-4, but little or no IL-5,
upon stimulation with PMA and ionomycin or immobilized anti-CD3
mAb. These cytokines are also produced by similar percentages of
peripheral blood T cells. The proportions of peripheral T cells
producing cytokines in response to PMA and ionomycin stimulation in our
study are similar to those in previous reports (34, 35).
In contrast to T cells and NT cells, both hepatic and peripheral NK
cells produce TNF- and IFN- only. Pharmacological stimulation of
both hepatic and peripheral T cells with PMA and ionomycin was
generally more potent than CD3 cross-linking as an activator of
cytokine production and resulted in greater numbers of cells expressing
all cytokines. This observation has previously been reported for T cell
clones (36), and the more complete sustained stimulation
by PMA and ionomycin is likely to reflect its independence of the
levels of cell surface CD3/TCR expression and/or the involvement of
other non-CD3-mediated signaling pathways. The percentages of hepatic
NT cells expressing cytokines were similar or slightly higher than the
percentages of hepatic NK cell and T cell fractions, and the majority
of HL produced IFN- and TNF-, about one-half of the T and NT
cells produced IL-2, and up to 15% produced IL-4. These results
indicate that the majority, though not all, of the lymphocytes in the
normal human liver produce cytokines of the Th1/Tc1 profile, a finding
that is consistent with previous reports of proinflammatory cytokine
production by hepatocytes and Kupffer cells (32, 37, 38)
and our demonstration of large numbers of cytotoxic lymphocytes in the
liver. To our knowledge, this is the first report of cytokine
production at the single cell level by HL subsets. The predominant
inflammatory pattern of cytokine secretion found for human hepatic NT
cells contrasts with that of murine peripheral NKT cells expressing
NK1.1 receptors, which most notably produce large amounts of IL-4
(5, 7). These findings suggest that human hepatic NT cells
are either functionally distinct from murine peripheral
NK1.1+ T cells, they include only small numbers
of NK1.1+ T cell homologues, or they are
polarized by the liver to predominantly produce Th1/Tc1 cytokines
only.
Cytokine production by mature T cells is generally polarized to Th1/Tc1
or Th2/Tc2 profiles, which appear to be mutually exclusive and are
mutually inhibitory (39). The results of the present study
indicate, however, that a significant number of hepatic T cells (mean
6%) can simultaneously produce IFN- and IL-4. Dual Th1/Th2 cytokine
production has previously been reported for V24-JQ TCR-bearing
(15, 18) and (40) T cells. In the
present study, up to 11% (mean 5.1%) of hepatic
V24+ T cells and 1–3% hepatic
+ T cells could simultaneously produce
IFN- and IL-4 upon PMA and ionomycin stimulation. Since 6% of all
hepatic CD3+ cells can simultaneously produce
these cytokines, but only 2.7% of hepatic
CD3+ cells express the V24 TCR chain, it is
clear that other, yet undefined, Th0 cells exist. Although it has not
been directly demonstrated at the level of single cells, murine
NK1.1+ T cells can also secrete both IFN- and
IL-4 and are thought to be Th0 cells (5, 6, 7). In vitro and
in vivo studies have shown that the cytokine profiles of these cells
depend both on the nature of activating stimulus and on the nature of
the cytokines and other soluble factors in the local microenvironment.
Activation of NK1.1+ T cells by CD3 cross-linking
or with CD1 results in the production of both IFN- and IL-4, whereas
stimulation of NK1.1 results in the production of IFN- only
(6, 7, 10). IL-12 stimulates NK1.1+
T cells to produce IFN- and inhibits their production of IL-4
(7, 25, 27), while IL-4 production by these cells requires
IL-7 and is promoted by glucocorticoids (24, 26, 28).
Because of their similarities to NK1.1+ T cells,
it is likely that human hepatic NT cells will be similarly regulated.
One would therefore expect the microenvironment of the liver to have an
important influence on their functional properties. Our finding that
the majority of human HL taken freshly from their hepatic environment
mainly produce Th1/Tc1 cytokines, suggests that the liver, a primary
location of NT cell activity, provides a predominantly inflammatory
microenvironment. Interestingly, the majority (mean 82%) of hepatic T
cells that produced IL-4 also produced IFN-, indicating that most
IL-4-producing T cells in the liver are of the Th0 type, rather than
conventional Th2 cells.
The results of the present study indicate that the normal adult human
liver is a site of potent immunological activity with multiple
cytotoxic functions and patterns of cytokine secretion. Crucial to this
activity is the presence of uniquely high numbers of NT cells in the
liver. A role for NT cells as effectors in innate immune responses is
suggested by their ability to respond rapidly to various Ag-nonspecific
stimuli and to spontaneously lyse target cells. A second innate immune
activity may lie in the Ag specificities of their TCRs. Hepatic NT
cells include V24-JQ TCR-bearing cells that recognize
glycosylceramide Ags presented by CD1d (10, 16, 17).
Hepatic NT cells are also particularly rich in
TCR+ cells, a subset of which have been
implicated in CD1c-restricted responses (41, 42).
Furthermore, other human CD1-restricted T cells, which are known to
express the CD8+ or
CD4-CD8- phenotypes that
are predominant among hepatic NT cells, and to recognize glycolipid
components of bacterial cell walls (43, 44), may be also
present in the NT cell fraction. Thus, the liver may have a role in
immune responses to lipid Ags that are relatively fixed in structure
and would constitute targets of innate recognition. Hepatic NT cells
are also likely to function at the interface between the innate and
acquired immune systems. Their ability to rapidly produce large amounts
of both Th1 and Th2 cytokines upon primary stimulation suggests that
they may regulate secondary adaptive immune responses. Defective
regulation of the Th1/Th2 balance by these NT cells is thought to have
a role in immune-mediated diseases (45, 46, 47).
Hepatic NT cells may also have a role in the destruction of autologous
cells. The liver has been postulated to be a site for the elimination
of activated T cells by apoptosis. Huang et al. (48, 49)
reported that, following activation of peripheral
CD8+ T cells with Ag, a down-regulation of TCR
expression is associated with their disappearance from the lymph nodes
and spleen to the liver, where they undergo apoptosis. The mechanism of
apoptotic death could be through the interaction of Fas (CD95, APO-1)
with its ligand (FasL), since lpr and gld mice
(which are deficient in Fas and FasL, respectively) accumulate large
numbers of CD4-CD8-
ßTCR+ T cells expressing low TCR levels in
the liver (50). Our findings of the liver being a site of
potent cytotoxicity are consistent with this hypothesis and suggest
that the liver might provide an "inflammatory" environment, as
observed in the present study, that induces the Ag-nonspecific
cytotoxic functions (LAK activity) of NT cells. Hepatic NT cells might
also lyse autologous cells via TCR ligation. CD1-restricted T cells
(15, 41) and TCR+ T cells
(51) can recognize and respond to target molecules in the
absence of any identifiable cognate Ag. Thus, the up-regulation of CD1
or heat shock protein expression by moribund target cells under certain
conditions, such as the microenvironment of the liver, might render
them susceptible to lysis by V24JQ, , or other NT cells. An
important function of such autoreactivity is the cytolytic destruction
of tumors, and several studies have implicated murine NKT cells
expressing the V14J281 TCR (21, 22) and human hepatic
CD3+CD56+ NT cells
(52) as important antitumor effector cells. Hepatic NT
cells are therefore likely to be future targets for manipulation in the
treatment of malignancies and immune-mediated diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Derek G. Doherty, Education and Research Centre, St. Vincent’s Hospital, Elm Park, Dublin 4, Ireland. E-mail address:
3 Current address: Institute of Liver Studies, King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, U.K.
4 Current address: Infection and Immunology Group, Biology Department, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
5 J.E.H. and C.O. contributed equally to the direction of this work.
6 Abbreviations used in this paper: KIR, killer inhibitory receptor; NT cell, natural T cell; MNC, mononuclear cells; HL, hepatic lymphocyte; LAK, lymphokine-activated killing; PerCP, peridin chlorophyll protein.
Received for publication March 16, 1999. Accepted for publication June 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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