Michael Owens
11/1/12
BIOL 303
Tumor Growth Factor-β1 Negatively Effects Tumor Surveillance by Natural Killer
Cells
Natural killer (NK) cells are cytotoxic lymphocytes integral to mammalian
tumor surveillance. The typical method of NK cell activation involves the
transmembrane receptor protein NKG2D (Fig. 1). While healthy cells have virtually
no NKG2D ligands (“activators”) on their surfaces, tumorous cells typically possess a
large quantity of these ligands (Guerra et al. 2008). In this study, the scientists bred
a line of NKG2D deficient mice. Compared to the control group, the deficient mice
developed much larger tumors. Also studied was the expression of NKG2D ligands
by tumors as compared with their ages. The results showed that tumors that arose
early during tumorigenesis showed an increased expression of NKG2D ligands
compared to late arising tumors. These results together supported the theory that
the NKG2D receptor aids in tumor surveillance by activating NK cells in the
presence of tumors (Guerra et al. 2008).
Tumor surveillance, though, is not always one hundred percent effective. To
determine why this is, the research team of Lee et al. (2004) began looking into the
possible causes of NKG2D down-regulation. Past research had discovered that
tumor growth factor-β1 (TGF-β1) was present in higher doses in cancer patients
than in healthy patients and that the greater the quantity of the growth factor, the
greater the progression of the tumor (Kong et al. 1996). Prior studies also showed
that TGF-β1 also inhibited in vitro NK cell cytotoxicity (Bellone et al. 1995). Taking
these facts together, Lee et al. (2004) hypothesized that TGF-β1 inhibited NK cell
function by down-regulating NKG2D.
To test this hypothesis, the researchers took blood samples from a total of
thirty-seven cancer patients and measured NKG2D and TGF-β1 levels. The results
showed a negative correlation between the levels of TGF-β1 and NKG2D (Fig. 2).
Next, to confirm that the growth factor did indeed down-regulate NKG2D, healthy
NK cells were incubated in TGF-β1 rich (cancerous) serum. In these cells, NKG2D
became under-expressed after serum introduction (Fig. 3.A, Lane 2). After this, antiTGF-β1 (meant to make TGF-β1 ineffective) was introduced into the serum, and an
increase in NKG2D levels was seen (Fig. 3.A, Lane 3). Two other tests (Fig. 3.A, Lanes
4-5) were also carried out to neutralize the cytokines interleukin-4 (IL-4) and
interleukin-10 (IL-10) in the serum to make sure only TGF-β1 was effecting NKG2D
levels; these tests confirmed that only TGF-β1 effected NKG2D regulation (in this
model). Further controls were also carried out by incubating healthy NK cells in
normal, non-cancerous serum. One group of cells received anti-TGF-β1 while others
received anti-IL-4 or anti-IL-10. NKG2D expression was identically among all of
these groups. All of these data together suggested that TGF-β1 down-regulates
NKG2D (Lee et al. 2004).
The next test carried out by Lee et al. was to determine if regulation by TGFβ1 was dose-dependent. Three different variants of this were carried out. The first
involved incubating non-active NK cells in various doses of TGF-β1 (Fig. 4, Column
1), while the second and third involved incubating two batches of cells that had been
activated by two different ligands (IL-2 and IL-15) (Fig. 4, Columns 2-3). The team
determined that NKG2D inhibition began at 0.1 ng/mL TGF-β1 and steadily
increased with progressively larger doses, with expression peaking at 5 ng/mL TGFβ1. A further test was carried out to confirm that TGF-β1 only regulated NKG2D. The
quantities of three different receptors (CD94, NKG2A, and CD44) were measured in
IL-2 activated NK cells and compared to these same cells introduced to TGF-β1. The
results showed no significant difference between the two (Fig. 5). These results
confirmed that TGF-β1 only regulates NKG2D (Lee et al. 2004).
The final tests carried out by Lee et al. aimed to discover what effects TGF-β1
had on NK cell cytotoxicity. In the first test, three NK cell variants (non-activated, IL2 activated, and IL-15 activated) were introduced to cells marked for lysis (CEM
cells) in various concentration of TGF-β1. The results (Fig. 6) showed that the
specific lysis of the NK cells was lower at higher concentrations of TGF-β1 for all NK
cell variants. The last test carried out was to determine if the effects of TGF-β1 on
cytotoxicity had any effect on apoptosis. In this test, the levels of two cytotoxic
elements (perforin and Fas) were measured in NK cells repressed by TGF-β1. The
results (Fig. 7.A) showed that the concentration of TGF-β1 had no effect on perforin
or Fas levels. In addition, it was determine that TGF-β1 did not induce NK cell
apoptosis by comparing the concentration of cytotoxic elements within normal and
TGF-β1-weakened cells (Fig. 7.B). All of these results together supported the initial
hypothesis that the down-regulation of NKG2D is caused by TGF-β1 (Lee et al.
2004).
The aforementioned papers together inspired the work of Espinoza et al.
(2012). The researchers hypothesized that TGF-β regulates NKG2D production by
up-regulating a microRNA (miRNA), specifically miRNA-1245. Using the algorithm
developed by Lewis et al. (2003), the team determined that miRNA-1245 can attach
to the 3’-untranslated region (3’UTR) of the NKG2D gene (see Fig. 8.A and 8.B for
more details). The team determined (through real time PCR) that miRNA-1245 is
expressed in both ex vivo (“fresh”) and cultured NK cells (Fig. 8.C). The team also
used the discovery that serum from cancer patients contained functional miRNA
(Turchinovich et al. 2011) to justify testing the serum of seven healthy individuals,
seven leukemia patients, and three myelodysplastic syndrome patients. The results
(Fig. 8.D) showed that none of the healthy individuals had a significant amount of
the miRNA-1245, that three of the seven leukemia patients had a significant
quantity, and that two of the three myelodysplastic syndrome patients had a
significant quantity. These facts together pointed towards the potential
effectiveness of this miRNA in the down-regulation of the NKG2D protein (Espinoza
et al. 2012).
Next, tests were done to confirm the nature of miRNA-1245’s interaction
with the 3’UTR of the NKG2D gene. Espinoza et al. engineered a vector with a
luciferase gene (NKG2D-3’ UTR-Luc) that contained the section of the 3’UTR to
which miRNA-1245 binds. A second vector was also engineered that contained a
luciferase gene that was missing the part of the 3’-UTR which contained the binding
site for miRNA-1245 (3’UTR-mut) to act as a control. Using both the NKL and HEK
cell lines, derivatives were grown that over-expressed miRNA-1245. Four groups of
each cell line were tested. One group consisted of wild types that received the
NKG2D-3’ UTR-Luc vector, the second consisted of wild types that received the
3’UTR-mut vector, the third consisted of over-expressers that received the NKG2D3’ UTR-Luc vector, and the fourth consisted of over-expressers that received the
3’UTR-mut vector. The over-expressers with NKG2D-3’ UTR-Luc had a much lower
expression of the luciferase than the wild type cells. Also, in both cell lines, the
expression of luciferase in the 3’UTR-mut cells was equal between the wild type and
over-expressers (Fig. 9). These results confirmed that miRNA-1245 binds to the
3’UTR of the NKG2D gene, as proposed by Espinoza et al. (2012).
The next phase of research was to determine what could cause the upregulation of miRNA-1245. The team found that both ex vivo (Fig. 10.A) and cultured
(Fig. 10.B) NK cells expressed much higher levels of pri-miRNA-1245 (recently
transcribed, double stranded miRNA) than mature miRNA-1245 (single stranded,
cut from pri-miRNA). This led the Espinoza et al. to hypothesize that the substance
that up-regulates miRNA-1245 must do so by catalyzing the transition between the
pri and mature forms. Using the research of Davis et al. (2008), the team decided
that TGF-β1 might be an inhibitory molecule, because it both up-regulated other
miRNA and down-regulated NKG2D. To test this, the team incubated ex vivo NK cells
with various doses of TGF-β1 and measured the level of mature miRNA through real
time PCR. The results showed a positive correlation between TGF-β1 levels and
mature miRNA-1245 levels, but no correlation between TGF-β1 levels and primiRNA-1245 levels (Fig. 10.C). Similar results were found when cultured NK cell
lines were incubated in TGF-β1 (Fig. 10.D). In addition, the team measured levels of
both mature and pri-miRNA-1245 over time after starting TGF-β1 treatment. While
the pri-miRNA-1245 levels remained constant, the levels of mature miRNA-1245
peaked after sixteen hours (Fig. 10.E). While the peaking expression of the mature
miRNA suggested that TGF-β1 very effectively modifies pri-miRNA-1245 into
mature miRNA-1245, the constant levels of the pri-miRNA also suggested that TGFβ1 slightly increased the level of miRNA transcription (Espinoza et al. 2012).
To test the effect of miRNA-1245 on NKG2D, Espinoza et al. infected ex vivo
NK cells with either a miRNA-1245 increasing vector (miRNA-1245-Vector) or a
control vector (which had no effect on the cell). Cells infected at a MOI (multiplicity
of infection) of 50 expressed as much miRNA-1245 as cells incubated with TGF-β1.
The cells infected with miRNA-1245-Vector showed a constant down-regulation of
NKG2D (Fig. 11.A and 11.B). Also measured were the levels of the mRNA transcripts
of NKG2D, which showed a similar trend (Fig. 11.C). A test for functionality of these
infected cells was then carried out. The infected cells were incubated in a plate
containing NKG2D ligands and the control cells were incubated on a plate of mouse
IgG ligands (meant to have no effect). The levels of tumor necrosis factor-α (a major
cytotoxic element in NK cells) were measured in both kinds of cells. The results (Fig.
11.D) showed that the control cells released significantly more tumor necrosis
factor-α than infected cells (Espinoza et al. 2012). These results showed that
miRNA-1245 is an effective down-regulator of NK cell cytotoxicity.
To add extra validity to the above results, Espinoza et al. (2012) established a
cultured cell line of miRNA-1245 knockout cells (miRNA-1245 5KO) through
infection by a vector. These cells expressed no miRNA-1245, in contrast to ex vivo
and control vector-infected cells. These cells also expressed more NKG2D protein
(Fig. 12.A) and more NKG2D mRNA transcripts (Fig. 12.B). When introduced to TGFβ1, the infected cells still slight showed down regulation of NKG2D, but ex vivo cells
showed significant greater down-regulation (Fig. 12.C). To further add to their
findings, the team infected ex vivo cells with either a vector that actively degraded
miRNA-1245 (antago-miRNA-1245) or a control (antago-NC). The antago-miRNA1245 cells showed greater levels of NKG2D and were less susceptible to TGF-β1
treatment (Fig. 12.D) than the control (Fig. 12.E) (Espinoza et al. 2012).
Taken together, the results of all the above research support that TGF-β1 is
an effective inhibitor of tumor surveillance by NK cells. Further topics of research
could possibly include the method in which miRNA-1245 down-regulated NKG2D
and the effects of other miRNAs on other immune cells. Also, because miRNA-1245
knockout cells still responded to TGF-β1, further research could be done into
possible other methods of regulation by this molecule.
Figures
Figure 1 (original)
Figure 2 (Lee et al. 2004)
The correlation between plasma TGF-β1 and NKG2D expression level in cancer patients was
determined by Pearson’s correlation coefficient (r), and the associated probability (p) were calculated
for each combination.
Figure 3 (Lee et al. 2004)
Incubation of NK cells with plasma obtained from cancer patients inhibits surface NKG2D expression
in a TGF-β1-dependent manner. Purified NK cells were cultured with 100 U/ml IL-2 in the presence of
a 1/5 dilution of plasma from a cancer patient (A) or a normal volunteer (B). Cells were harvested after
24 h and stained with anti-NKG2D mAb. Single-color flow cytometry was performed, and results are
presented as the fold change in MFI. For neutralization experiments, 10 μg/ml mAb against TGF-β1,
IL-4, or IL-10 was added to the culture.
Figure 4 (Lee et al. 2004)
Effect of TGF-β1 on NKG2D expression of freshly isolated or lymphokine-activated human NK
cells. Purified NK cells were cultured with or without 100 U/ml IL-2 or 100 ng/ml IL-15 in the presence
of the indicated concentrations (nanograms per milliliter) of TGF-β1. Cells were harvested after 2 days
and were stained with specific mAb for NKG2D. Single-color flow cytometry was performed (x-axis,
log10 fluorescent intensity; y-axis, cell count). Solid and dotted lines indicate staining with anti-NKG2D
mAb and its isotype-matched control, respectively.
Figure 5 (Lee et al. 2004)
TGF-β1 does not affect surface expression of CD94/NKG2A or CD44 on IL-2-activated human NK
cells. Purified NK cells were cultured with 100 U/ml IL-2 in the presence or the absence of 5 ng/ml
TGF-β1. Cells were harvested after 2 days and were stained with specific mAbs for each NK receptor.
Single-color flow cytometry was performed (x-axis, log10 fluorescent intensity; y-axis, cell count). Solid
and dotted lines indicate staining with anti-CD94, anti-NKG2A or anti-CD44 mAb and their isotypematched controls, respectively.
Figure 6 (Lee et al. 2004)
TGF-β1 inhibits the cytotoxicity of freshly isolated or lymphokine-activated human NK cells. Purified
NK cells were cultured in the presence or the absence of 100 U/ml IL-2, 100 ng/ml IL-15, or the
indicated concentrations (nanograms per milliliter) of TGF-β1. Cells were harvested after 2 days and
subjected to 4-h 51Cr release assays using CEM target cells. Results are expressed as the percentage
of specific lysis.
Figure 7 (Lee et al. 2004)
TGF-β1 does not affect the level of perforin, Fas ligand, annexin V, or PI in freshly isolated or
lymphokine-activated human NK cells. A, Purified NK cells were cultured in the presence or the
absence of 100 U/ml IL-2, 100 ng/ml IL-15, or 5 ng/ml TGF-β1 for 2 days. Cells were harvested,
permeabilized, stained with anti-perforin or anti-Fas ligand mAb, and analyzed by FACS (x-axis,
log10 fluorescent intensity; y-axis, cell count). The dotted lines are negative controls; the solid lines are
TGF-β1-untreated cells; the bold lines are TGF-β1-treated cells. B, Flow cytometric analysis using
annexin V and PI was conducted on NK cells activated as described in A.
Figure 8 (Espinoza et al. 2012)
A
A schematic representation of the interaction between miR-1245 and its target site in
the 3′UTR region of NKG2D mRNA.
Computational modeling showed the hybridization of miR-1245 and the 3′UTR region
of NKG2D mRNA; mfe represents the calculated minimal free energy
C
Mature miR-1245 expression in fresh or activated primary NK cells was measured by
quantitative RT-PCR and normalized to U6B RNA. The data are the means ± S.E.M. (n=
13).
D
The expression of mature miR-1245 in exosomes isolated from plasma samples from
patients with non-Hodgkin’s lymhoma (NHL) (n=7), acute myelogenous
leukemia/myelodysplastic syndrome (AML/MDS) (n=3) and from seven healthy donors.
Figure 9 (Espinoza et al. 2012)
The interaction of miR-1245 with the NKG2D gene 3′UTR. (A) NKL cells or (B) HEK cells
overexpressing miR-1245, or their wild type (WT) counterparts, were transfected with a
luciferase expression vector (pGL3-TK-Luc), or with constructs including
theNKG2D 3′UTR (NKG2D-3′ UTR-Luc) or with a luciferase expression vector that
included the NKG2D 3′UTR with a 16-bp deletion of the miR-1245 targeting site (3′
UTR-mut). The firefly luciferase activities measured 48 h after transfection were
normalized to the Renilla luciferase expression and the mean activities ± S.E.M. from
three independent experiments are shown.
Figure 10 (Espinoza et al. 2012)
The expression of the primary gene transcripts (pri-miR-1245) in fresh and cultured NK
cells normalized to GAPDH. The data are the mean values ± S.E.M. from measurements
in seven donors.
The expression of pri-miR-1245 normalized to GAPDH in the NK cell lines YT, NK92,
KYHG-1, and NKL.
The expression of pri-miR-1245 and mature miR-1245 in primary NK cells treated with
various concentrations of TGF-β1 for 24 h (n=7).
The expression of mature miR-1245 in NK cell lines after treatment with TGF-β1 (2.5 or
10 ng/mL) for 24 h. The error bars in (C) and (D) show the ± S.E.M. from three
independent experiments, each measured in duplicate.
he time course of pri-miR-1245 and mature miR-1245 expression in primary NK cells
after treatment with TGF-β1 (10 ng/mL) (n=7). The data are the means ± S.E.M. from
three independent experiments, each measured in duplicate.
Figure 11 (Espinoza et al. 2012)
A representative result showing the cell surface expression of NKG2D on miR-1245transduced primary NK cells. Cells stained with anti-NKG2D (filled histograms) or with
the isotype antibody (open histogram).
The summarized data from 12 donors are shown.
Fresh NK cells were transduced as in the panels A and B, and the NKG2D mRNA levels
were measured by quantitative RT-PCR 24 h after transduction. The relative results
normalized to U6b RNA are expressed as the percentage of the mRNA compared to that
for the control conditions (untreated cells).
Fresh NK cells transduced with the miR-1245-vector or with the NC-vector (n=7) and
cultured in plates coated with a mixture of three recombinant NKG2D-Ls (MICA, ULBP-1
and ULBP-2) or control IgG.
Figure 12 (Espinoza et al. 2012)
KYHG-1 cells were examined by flow cytometry for NKG2D expression. The filled
histogram represents shRNA-NC-transduced KHYG-1 cells stained with anti-NKG2D
antibody. In the open histogram, the solid line shows the data from the miR-1245 5KO
KHYG-1 cells stained with the anti-NKG2D antibody, and in the open histogram, the
dotted line indicates cells stained with the isotype antibody. A representative figure from
three independent experiments is shown.
Total RNA was extracted, and the levels ofNKG2D mRNA normalized to the levels
of GAPDH mRNA were measured by RT-PCR. The figure shows the means ± S.E.M. from
three independent experiments.
miR-1245 5KO KHYG-1 cells or wild-type (WT) KHYG-1 cells were cultured for 48 h in
the presence or absence of TGF-β1, and their NKG2D expression levels were examined
by flow cytometry. The filled histogram represents untreated cells, the green histograms
represent cells treated with TGF-β1 (5 ng/mL), the red histograms represent cells
treated with TGF-β1 (10 ng/mL) and the blue histograms with the dotted line represent
cells stained with the isotype antibody. A representative figure from the data obtained
from three independent experiments is shown.
Cultured NK cells were left untreated or were transfected with anta-go-miR-1245 or
antago-NC, and 48 h later, their NKG2D expression levels were examined by flow
cytometry. The filled histogram represents cells transfected with antago-miR-1245 or
antago-NC, the open histogram with solid lines represents untreated cells, and the open
histograms with the dotted line represent cells stained with the isotype antibody. A
representative figure from the data obtained from three independent experiments is
shown.
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