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PLENARY PAPER
From the Department of Medicine, Division of Bone
Marrow Transplantation, Stanford University Medical Center, Stanford,
CA.
T cells with natural killer cell phenotype and function (NKT cells)
have been described in both human and murine tissues. In this study,
culture conditions were developed that resulted in the expansion of
CD8+ NKT cells from bone marrow, thymus, and spleen by the
timed addition of interferon- T cells with natural killer (NK) cell
activity have been identified in both murine and human
tissues.1-4 Murine NKT cells typically express
phenotypic markers found on T cells such as CD3 and the Recently, a second population of NKT cells has been described that
expresses a variable TCR repertoire and is not dependent on CD1d for
maturation and development.14 In addition, these CD1d-independent splenic NKT cells expressed mainly CD8 or were double
negative with respect to CD4 and CD8 expression. Approximately 1% to
2% of splenocytes from C57BL/6 mice are
T cells are readily expandable following mitogenic stimulation
with monoclonal antibodies (MAbs) directed against the TCR complex.15 The combination of anti-CD3 and IL-2 results in
the expansion of murine and human T cells capable of lysing a variety of different tumor cell lines, some of which are resistant to NK
cells.16-18 The expanded cells also have in vivo antitumor
cell activity against murine tumors16,19 and human tumors
transplanted into immunodeficient mice.18,20 It has been
demonstrated that T cells activated with anti-CD3 and IL-2 and cultured
for 6 to 8 days have a decreased capacity for inducing
graft-versus-host disease (GVHD). Both CD4+ and
CD8+ cells were less likely to result in GVHD following
activation with anti-CD3 and IL-2.21
In this report we have used culture conditions that allow for the
growth of large numbers of T cells that share functional and phenotypic
properties with NK cells. Following activation and culture,
Animals
Expansion of NKT cells
Flow cytometry The following MAbs were used in this study. MAbs against CD3, CD4,  TCR,   TCR, CD8, CD25, CD69, Ly49D, and Ly49G2 were
conjugated to fluorescein isothiocyanate (FITC). MAbs against CD3, CD4,
CD8, NK1.1, DX5, Ly49A, and Ly49C were conjugated to phycoerythrin (PE;
Pharmingen, San Diego, CA). PE-conjugated mouse immunoglobulin (Ig)G2a , rat IgG2a, hamster IgG, and FITC-conjugated mouse
IgG2a, rat IgG2a, and rat IgM were used as negative isotype
controls. Approximately 106 cells were incubated with Fc
Block (antimouse CD16/CD32 MAb; Pharmingen) for 30 minutes at 4°C
followed by specific antibodies. Excess antibody was removed by
washing, and the stained cells were analyzed, using a FACSscan (Becton
Dickinson, San Jose, CA). In some experiments,
CD3+NK1.1+ and
CD3+NK1.1 or CD3+DX5+
and CD3+DX5 populations of cells were
isolated by flow cytometry, using a fluorescence-activated cell sorter
(FACS) Star Sorter.
51Cr release cytotoxicity assay Target cells, Bcl1 (B-cell lymphoma H-2d), P3x63 AG8U.1 (myeloma H-2d), C6VL (leukemia H-2b), EL4 (T-cell leukemia H-2b), P815 (mastocytoma H-2d), and YAC-1 (T-cell lymphoma H-2a) were labeled with 51Cr (Dupont-NEN, Boston, MA) by incubating 1-2 × 106 cells in 300 µCi 51Cr for 2 hours at 37°C. The labeled cells were washed with phosphate-buffered saline 3 times, then distributed in 96-well plates at 2 × 104 cells/well in triplicate. Effector cells were added at the indicated ratio and incubated for 4 hours at 37°C. The cells were then pelleted by centrifugation, and aliquots of supernatant were counted in a gamma counter. The percentage of specific 51Cr release was calculated according to the following formula: % specific lysis = (test release) (spontaneous
release) × 100/(maximal release) (spontaneous release).
Spontaneous release was obtained by incubating cells in media alone,
and maximal release was obtained with 2% Nonidet-P40 incubation.
Cytokine production Splenocytes (2 × 105) or expanded NKT cells at 0, 7, 14, and 21 days of culture were removed, washed, and incubated with 5 µg/mL phytohemagglutinin (PHA; Sigma Biochemicals, St. Louis, MO) or media alone in a total volume of 200 µL/well in a 96-well plate and cultured for 48 hours. The supernatant was harvested and assayed for cytokine production by enzyme-linked immunosorbent assay (ELISA) for IFN-, IL-4, IL-10, and tumor necrosis factor (TNF-;
Biosource, Camarino, CA) according to manufacturer's instructions.
Cytokine production was also evaluated at the same time points by RNase
protection assay (Pharmingen, San Diego, CA) according to the
manufacturer's instructions.
In vivo experiments To establish malignant disease, 6- to 8-week-old Balb/c recipient mice were first injected with 103 Bcl1 leukemic cells intraperitoneally. Tumors were allowed to grow for 7 days, and then mice were irradiated with 8 cGy in 2 split fractions. On the same day, animals were rescued with 1 × 107 BM cells, and some animals received 2-4 × 107 expanded CD8+ NKT cells by intravenous injection derived from syngeneic Balb/c donor animals. Animals were observed for signs or symptoms of tumor growth (massive splenomegaly), and time from cell injection to animal death was monitored as previously described.22 The cause of death was determined by necropsy.To explore the capacity of the CD8+ NKT cells to cause GVHD, recipient Balb/c mice were irradiated as above and transplanted with BM cells from C57BL/6 recipients with either splenocytes or expanded CD8+ NKT cells from wild-type (WT) C57BL/6 or mutant strains of mice on the B6 background at the indicated dose. Typical signs and symptoms of GVHD were observed, including ruffled fur, diarrhea, hunchback appearance, weight loss, and survival. Statistical methods Cytotoxicity of sorted populations of cells was evaluated, using the Student t test. Animal survival was compared, using the log-rank test.
Expansion of NKT cells Cells were expanded from spleen, thymus, and BM from different strains of mice by incubating the cells in the presence of murine recombinant IFN-. One day later, the cells were transferred to a
coated flask with MAb against CD3, and media were supplemented with
IL-2. Expansion of cells from the spleen, thymus, and BM were assessed
at regular intervals for proliferation and viability (Figure
1). Under these culture conditions, cells
expanded rapidly from the spleen up to 200-fold by day 21. Total cell
expansion was less dramatic from the thymus and BM. These results
reflect the relatively low numbers of CD8+ cells in these
tissues, especially in the BM. If purified CD8+ cells are
isolated from these tissues, they readily expand similar to that of
splenocytes (data not shown). Cells from all tissues cultured with IL-2
alone did not expand and were not viable after 7 days in culture. Cells
cultured with IL-2 and IFN- without CD3 MAb maintained their
viability until day 14 but did not expand. Cell expansion from
splenocytes was similar from C57BL/6, Balb/c, as well as the knock-out
(KO) strains of mice (data not shown).
Phenotype of the expanded cells FACS analysis was performed on cultures initiated from the different tissues on day 0 and every 7 days thereafter. The percentages of lymphocytes expressing CD3, CD8, CD4,TCR, TCR, CD69,
CD25, CD44, B220, DX5, NK1.1, or Ly49 molecules were determined at each time point. The phenotype of the cells derived from spleen over a
21-day period is shown in Figure 2. At
the initiation of culture, the phenotype of the splenocytes were
typical of naive unmanipulated cells with approximately 24%
CD3+, 8% CD8+, 15% CD4+, and 20%
TCR+ (Figure 2A). These cells did not express the
activation markers CD69 or CD25 but were CD44+ (Figure 2B).
A small population (5%) of cells expressed the NK markers NK1.1 and
DX5 (Figure 2C) with the majority of these cells being
CD3 (data not shown). By day 7, there was a dramatic
increase in the percentage of
CD3+CD8+TCR+ cells. Cells
were negative for both myeloid (Gr-1) and monocytic (Mac1) markers
(data not shown). By day 21 of culture, virtually all of the cells were
CD3+CD8+TCR+ with a
significant percentage (20% to 50%) of the cells co-expressing the NK
cell markers NK1.1 and DX5 (Figure 2A,C). A similar phenotype was
observed from cells expanded from thymus and BM. Cells expressing TCR were relatively rare (1% to 6%) at the initiation of
culture and were no longer detectable after 21 days of culture. The
phenotype of the majority (> 90%) of cells after 14 to 21 days in
culture from all tissue sources (BM, thymus, and spleen) was similar
and found to be CD3+, CD8+, or
TCR+ (data not shown). The expanded cells were highly
activated as assessed by CD25 and CD69 expression (Figure 2B).
Approximately 4% to 5% of splenocytes expressed Ly49A, C, D, or G2.
However, after day 7 of culture, very few cells (< 2%) expressed
these markers (data not shown). Morphologically, the expanded cells were large, granular, and vacuolated. Evaluation of TCR repertoire using a panel of V MAbs showed a varied TCR usage that does not significantly change over time in culture, indicating that the expanded
cells are polyclonal (data not shown). A number of functional studies
were performed with these expanded cells.
Cytotoxicity against syngeneic and allogeneic tumor cell lines Splenocytes had no cytolytic activity on day 0. However, by day 7, significant lytic activity was observed against P3x63 tumor cells with up to 65% specific lysis at an effector-to-target (E:T) ratio of 40:1. By day 14 to day 21, the CD8+ NKT cells exhibited more than 80% specific lysis against this tumor cell line at a 40:1 ratio (Figure 3A). The expanded CD8+ NKT cells maintained their cytolytic activity for up to 60 days in culture (data not shown). Cytotoxicity of the expanded CD8+ NKT cells against different tumor cell lines was assessed by a 4-hour 51Cr release assay. A variety of different tumor cell lines were used, including P815 (mastocytoma, H-2d), YAC-1 (T-cell lymphoma, H-2a), P3x63 (myeloma, H-2d), Bcl1 (mouse B-cell lymphoma, H-2d), EL4 (lymphoma, H-2b), and C6VL (thymoma, H-2b). The representative results are shown in Figure 3B, using effector cells derived from C57BL/6 mice. The expanded CD8+ NKT cells were capable of recognizing syngeneic (EL4) and allogeneic (P815, P3 × 63, Bcl1, and YAC1) targets. Interestingly, C6VL tumor cells were consistently resistant to cytolysis. A similar pattern was observed when CD8+ NKT cells were expanded from Balb/c splenocytes, where, again, all target cells except C6VL were sensitive to cytolysis. Similar cytotoxicity results were obtained, using cells derived from the thymus and BM as compared to splenocytes from B10.D2/nSnJ and FVB/J animals (data not shown), indicating that expansion of this population of cells is not strain specific. In contrast, when freshly isolated normal cells derived from BM or spleen were used as target cells, no cytolytic activity was observed (data not shown).
To better define the population of cells with cytolytic activity, the
expanded CD8+ NKT cells were sorted into
CD3+DX5+ and CD3+DX5
Cytokine production Cytokine production by the expanded CD8+ NKT cells was assessed for the production of IFN-, TNF-, IL-4, and IL-10 by
ELISA. To perform these studies, splenocytes were expanded under the culture conditions described above and harvested at weekly intervals. The harvested cells were evaluated for cytokine production with and
without activation in vitro for 24 hours with PHA. Supernatants were
harvested and assayed by ELISA to quantitate cytokine production. The
major cytokine produced by the expanded CD8+ NKT cells was
IFN-. Levels of IFN- produced by the CD8+ NKT cells
increased over time in culture (Figure
4A). At day 0, low levels of IFN- were
found. However, by day 7, production of IFN- by the expanded cells
increased dramatically and by day 21 was approximately 6000 pg/mL with
or without PHA activation. TNF- production was similarly
up-regulated to approximately 1800 pg/mL by day 21. Moderate levels of
IL-10 (800 pg/mL) were produced mainly early in the culture period (day
7) that declined over time. In marked contrast, little or no IL-4
(10-20 pg/mL) was detected. Cytokine production was also evaluated by
RNA expression, using an RNase protection assay (Figure 4B). This assay
confirmed high levels of IFN- messenger RNA (mRNA) as well as low
levels of IL-10 and little to no IL-4 mRNA (data not shown). mRNA
expression of TNF-, TNF-, macrophage migration inhibitory factor,
and tumor growth factor were all up-regulated over time in culture.
No expression of IL-6 or IFN- was found at initiation of cultures or
at the end of expansion (Figure 4B).
Role of CD1d on expansion of CD8+/ / KO animals. At day 0 prior to expansion, the
phenotype of the starting population from CD1/
splenocytes was similar to WT with approximately 25% CD3+
T cells and a slightly higher percentages of CD4+ T cells
as compared to CD8+ T cells (Figure
5). Both animals had low numbers of
NK1.1+ cells with the majority of these cells being
CD3. Splenocytes from CD1/ animals had a
small population (approximately 3%) of
CD3+NK1.1+ cells. Following ex vivo expansion
under the culture conditions described above, both populations of
splenocytes expanded to a similar degree (data not shown). The
phenotype after 21 days in culture was also very similar with the
majority of cells expressing CD3, CD8, and TCR. Cells derived
from CD1d/ animals had a small percentage of
CD4+ cells following expansion. A significant percentage of
CD3+ T cells also expressed NK1.1 (19% to 30%) that was
similar in cells derived from either WT or CD1/
splenocytes (Figure 5). Cytotoxicity was also similar with both cultures of expanded cells displaying significant cytotoxicity against
a broad array of tumor cell targets. Moreover, using
CD1d/ targets that were transduced with the CD1d
molecule showed essentially no difference in cytotoxicity (data not
shown). These data strongly suggest that CD1d plays little to no role
in the expansion or cytotoxicity of the CD8+ NKT
cells.
In vivo antitumor effect of expanded NKT cells The in vivo antitumor activity of the expanded CD8+ NKT cells was studied in a syngeneic model system in which Balb/c mice were injected with an otherwise lethal dose of Bcl1 tumor cells. This tumor cell line results in massive splenomegaly and ultimately death of the recipient animals within approximately 2 months.22 In these studies, Balb/c mice were injected with 103 Bcl1 cells that were allowed to grow for 1 week to simulate active disease. The mice then received lethal irradiation to attempt to develop a state of minimal residual disease similar to the clinical situation. The recipient animals were then treated with an infusion of BM cells or BM plus a single injection of 2-4 × 107 expanded CD8+ NKT cells of Balb/c origin. No additional cytokines, such as IL-2, were used. A representative experiment is shown in Figure 6. Animals that received radiation alone without rescue with BM cells all died within 20 days of hematopoietic failure. Animals that received both Bcl1 tumor cells and BM survived the effects of radiation, but all died with progressive tumor growth within 55 days. The animals that received the expanded CD8+ NKT cells in addition to the BM and Bcl1 tumor cells had improved overall survival with approximately 50% of the animals surviving for more than 140 days (P = .004). These data demonstrate the in vivo antitumor activity of the expanded CD8+ NKT cells in a syngeneic model system. Further, these experiments demonstrate that CD8+ NKT cells do not interfere with hematopoietic engraftment.
CD8+/  Balb/c). In this system, BM cells alone do not
cause GVHD, and GVHD is induced with naive splenocytes. Injection of
2.5 × 106 unmanipulated splenocytes resulted in acute
GVHD and death within 20 days (Figure 7).
Interestingly, CD8+ NKT cells had a much lower propensity
for GVHD induction with little to no GVHD following injection of
5 × 106, 10 × 106, and even
20 × 106 cells (Figure 7). Although these animals did
experience mild weight loss (Figure 7A) as compared to animals that
were transplanted with BM alone, all of the animals survived (Figure
7B). The CD8+ NKT cells were not capable of preventing GVHD
by splenocytes (data not shown).
To explore the mechanism of action of the CD8+ NKT cells, a
series of animals deficient in key effector molecules were used, including fas deficient, fas ligand deficient, as well as KO animals lacking IL-2, IFN-
T cells with NK cell activity have been described in both murine and human tissues.1-4 NKT cells have generated considerable interest due to the ability of this population of cells to produce cytokines such as IL-4 and to possibly play a role in immune regulation and surveillance.10,11 To date, 2 populations of NKT cells have been described. One population of NKT cells is primarily CD4+ or lacks expression of either CD4 or CD8, co-expresses the NK marker NK1.1, and has a restricted TCR repertoire expression.5,6,23 This population of NKT cells is found primarily in the liver and thymus, recognizes CD1d and, on stimulation produces large quantities of IL-4.8,10 CD4+NK1.1+ cells have also been found to suppress GVHD.24 More recently, a second population of NKT cells has been described that is primarily CD8+, also co-expresses NK1.1, and has a more diverse and variable TCR repertoire.14 This population of NKT cells does not depend on CD1d and has been found primarily in the spleen and BM. Little is known about the biological function of these CD8+ NKT cells that are found at low frequency (approximately 1% to 3%) in the spleen and BM. A major advantage of T-cell populations is that they are readily
expandable on activation. In this report we have used similar cytokine
conditions that result in the expansion of both murine and human T
cells from peripheral blood.17,18,20 These conditions include the cytokines IFN- With the use of these culture conditions that favor T-cell expansion,
large numbers of CD8+ NKT cells can be readily expanded
that can be used for both in vitro and in vivo studies. CD1d is not
involved in expansion and cytotoxicity of these CD8+ NKT
cells, in contrast to the critical role of CD1d in the development and
function of CD4+ NKT cells.11,13 The lack of
CD1d involvement was demonstrated, using KO mice deficient in this
molecule. Cell expansion and phenotype of the cells were identical when
splenocytes from CD1d To explore the potential role of CD8+ NKT cells in vivo, experiments were performed in both the syngeneic and allogeneic setting. Expanded CD8+ NKT cells were capable of protecting a significant proportion of mice from an otherwise lethal challenge of Bcl1 lymphoma cells. In vivo activity was not dependent on IL-2 administration, and no toxicity was observed following the intravenous injection of up to 50 × 106 CD8+ NKT cells. Relatively large numbers of CD8+ NKT cells were used in the in vivo experiments; however, since the cells are readily expandable, this was easily achieved. In other studies, following stimulation with anti-CD3 and IL-2 and short-term culture in vivo, biological activity was also observed; however, IL-2 was required.16,27-29 Additional studies are under way to determine whether exogenous administration of cytokines such as IL-2 or IL-12 increases the in vivo activity of this cell population. To further explore the role of CD8+ NKT cells in the allogeneic transplantation setting, a number of studies were performed to determine whether this population of cells is capable of the induction of GVHD. Doses of between 5 and 20 × 106 CD8+ NKT cells were used that did not result in clinically significant GVHD. Animals that received CD8+ NKT cells had much improved survival in contrast to animals that received unmanipulated splenocytes, all of whom died within 20 days even at 10 times lower cell numbers. A different strain combination of B10.BR (H-2a) donor-activated T cells into Balb/c (H-2d) recipients also produced markedly reduced GVHD as compared to freshly isolated splenocytes.21 In other studies, NK cells have also been found to be capable of crossing histocompatibility barriers without causing GVHD.30 A number of hypotheses can be proposed to explain why CD8+ NKT cells cause much less GVHD than unmanipulated splenocytes. One possibility, as discussed above, is that GVHD in this model system is primarily caused by CD4+ T cells. However, this does not explain the observed results since freshly isolated CD8+ T cells (> 1 × 106) are capable of causing GVHD in this strain combination.31 In our experiments, up to 20 × 106 CD8+ NKT cells were injected with minimal GVHD. A second possibility is that activation of the T cells results in a change in the cells such that GVHD is attenuated. This could include the production of protective cytokines. In addition, activation of CD4+ T cells also results in attenuation of GVHD21 (and our unpublished observations). A third possibility is that the activated CD8+ NKT cells do not home properly. In fact, cultured T cells have been shown to home preferentially to the lungs and liver.21,32,33 Although homing was not directly assessed in this study, the CD8+ NKT cells were capable of protecting syngeneic and allogeneic recipients from a lethal dose of Bcl1 (this study and MR Verneris, submitted for publication). In fact, donor-derived CD8+ NKT cells could be found in the peripheral blood of recipient mice for at least 3 weeks following injection. Therefore, it seems unlikely that homing differences alone explain the observed results. A fourth possibility is that following allorecognition, previously activated T cells may undergo activation-induced cell death (AICD) that has been described for activation through the CD3/TCR complex.34,35 AICD is thought to proceed through fas ligand-fas interactions, although other molecules may also be important.36,37 To explore the mechanisms underlying the biological activity of the
CD8+ NKT cells, a number of different mouse strains lacking
key effector molecules were used. We studied CD8+ NKT cells
derived from fas ligand-defective, fas defective, and perforin, IL-2
and IFN- The role of IFN- The relationship of the expanded CD8+ NKT cells to that of
freshly isolated CD8+NK1.1+ cells requires
further evaluation. However, these studies are difficult to perform due
to the paucity of these cells in fresh tissues. NK1.1 expression may
represent an activation marker on T cells. This has recently been
demonstrated on virus-specific CD8+ and CD4+ T
cells40 and on We have performed similar studies with human peripheral blood
lymphocytes. Following 14 to 21 days in culture, the majority (> 90%) of cells are CD3+ with a significant percentage
of the cells co-expressing the human NK marker CD56.20,42
Ex vivo-expanded human CD3+CD56+ T cells
produce cytokines of the TH1 type and have broad
non-MHC-restricted cytotoxicity against a variety of tumor cell lines
as well as autologous and allogeneic fresh tumor
isolates.43,44 Stimulation with IFN- |
production
Top
Abstract
Introduction
, indicates spleen; 
,
BM; and 
, thymus.
indicates 1:5; 
, 1:10; 
, 1:20, and
indicates XRT (n = 5); 
, BM
(n = 5); and 
,
splenocytes; 
, NK 
, NK 
, NK 