2. Article 1: Introduction
Scientists have begun to examine the expression of certain immunological proteins associated with tumor
microenvironments and tumors themselves. It appears that some of these proteins negatively influence the
development and activation of T cells. One of the proteins under investigation is known as Programmed Death-1
(PD-1), which is a receptor found on the surface of circulating lymphocytes. The Ligands of PD-1 are Programmed
Death-Ligand 1 (PD-L1) and Programmed Death-Ligand 2 (PD-L2), both of which are expressed on a number of
different cell types and can be up regulated by pro-inflammatory cytokines. When PD-1 binds to either of its
ligands, the interaction causes either T cell inactivation or T cell exhaustion (Role of PD-L1 Pathway, 2015). It has
been noted that PD-L1 and PD-L2 are expressed on a variety of malignant tumor types and also on tumor-infiltrating
immune cells, allowing for the cancer cells’ continued growth. PD-L1 and PD-L2 expression in human chordomas is
yet to be examined (Mathios, D., et al. 2015). A chordoma is a rare form of cancerous tumor that originates from
what is believed to be the remnants of the notochord. The prognosis of those with chordomas is grim, with 67% of
survival rates being at 5 years or less. Furthermore, surgical resection and radiation are the only approved treatment
types for chordomas. Article 1, entitled “PD-1, PD-L1, PD-L2 expression in the chordoma microenvironment”,
analyzes the basal and inducible expression of the PD-1 Ligands in three different chordoma cell lines (Mathios, D.,
et al. 2015).
Methods
The chordoma cell lines used in this experiment were U-CH1, which was derived from a recurrent
radiation-treated sacrococcygeal chordoma, U-CH2, which was taken from a recurrent sacral tumor that had also
been treated by radiation, and JHC7, which was acquired from a surgically resected primary spinal chordoma.
Multiple tests were performed to determine expression of PD-1, PD-L1 and PD-L2 expressions on these cell lines,
before and after stimulation with specific cytokines. All tests for localization of proteins were performed in vitro, as
researchers still lack a competent mouse model for the study of chordomas. This represents a limit to the following
methods, as an in vitro response by cells may differ from an in vivo response. Flow cytometry was used to
determine the surface expression of PD-L1 and PD-L2 at the base level, after stimulation with interferon-gamma
(IFN-γ) and after stimulation with interleukin-4 (IL-4). These two cytokines were chosen to potentially induce PD-1
ligand expression in cells due to their antagonistic nature, as the functions of IL-4 are somewhat of a reversal of the
functions of IFN-γ. One experimentally relevant function of IFN-γ is its turning on of transcription factors that
3. produce pro-inflammatory cytokines like IL-1 and TNF-α , which was expected to induce PD-1 ligand expression
(Paludan, S. R. 1998). Cells from each line were treated with IFN-γ or IL-4, washed with PBS, then stained for PD-
L1 and PD-L2. To exclude dead cells from the flow cytometer readings, Aqua fixable viability dye was used. An
isotype control (cells not expressing PD-L1 or PD-L2) was used to gauge the levels of PD-L1 and PD-L2 staining,
and an LSR II flow cytometer was used to analyze cells before and after their cytokine treatments (Mathios, D., et
al. 2015). Real time-polymerize chain reaction (RT-PCR) was used to measure relative gene expression of PD-L1
and PD-L2 in two of the chordoma cell lines. RNA from the U-CH1 and U-CH2 cell lines was extracted and used to
synthesize 100µL of cDNA. The cDNAs from each of the two cell lines were combined separately with a master
mix, a primer mix, DEPC treated water, and most importantly, a prime mix for the gene of interest (PD-L1 gene or
PD-L2 gene). IL-4R was used as a control, as its fluorescence signal served as the background level with which to
measure the other fluorescence signals against, allowing for production of ΔCt values. A Ct (or cycle threshold)
value, is defined as the amount of times a cycle must be repeated in order for a fluorescent signal to exceed the
background level. The Ct value is indirectly proportional to the amount of target RNA present.
Immunohistochemistry was used to view local tissue expression of PD-1 and PD-L1. 5µm-thick sections of
chordoma samples were stained for PD-1 and PD-L1. Monoclonal antibodies specific for each protein were used in
each case as visible markers, as they would bind exclusively to either PD-1 or PD-L1. Tonsil tissue was used a
control for PD-1 staining, as it should express typical healthy levels of the target protein, and an IgG isotype control
was used for PD-L1 staining, as IgG is not specific for PD-L1. Tumor cells and tumor-infiltrating lymphocytes were
also scored for PD-L1 and PD-1 expression. Through visualization of the antibody markers used, localization and
threshold of membranous expression of both proteins was analyzed and scored by three certified pathologists
(Mathios, D., et al. 2015).
Results
The results of flow cytometry indicated that less than 5% of the cells tested expressed basal levels of PD-
L1 and PD-L2. However, stimulation by IFN-γ produced a statistically significant up regulation of PD-L1 in the U-
CH1 and JHC7 cell lines, and PD-L2 in all three of the cell lines. Stimulation with IL-4 did not produce a
statistically significant change in expression of PD-L1 or PD-L2 in any of the cell lines. Testing through RT-PCR
confirmed the expression of both PD-L1 and PD-L2 at the mRNA level in the U-CH1 and U-CH2 cell lines. Both of
these lines showed a significant rise in expression of the mRNA transcripts when stimulated with INF-γ. When
4. stimulated with IL-4, the expression of PD-L1, PD-L2 and IL-4 receptor transcripts was not affected in the U-CH1
cell line, but interestingly, all three transcripts were considerably up regulated in the U-CH2 cell line. Tumor-
infiltrating lymphocytes and macrophages were observed in 6 out of 10 of cases, and in half of these 6 cases, the
immune cells stained positive for PD-1 expression. Of the 6 cases, 4 stained positive for PD-L1 expression, which
was localized to areas in which the PD-1 expressing immune cells had infiltrated. Of all 10 cases, none of the tumor
cells demonstrated heavy expression of PD-L1 (Mathios, D., et al. 2015).
Discussion
Although little was known about the role and regulation of the PD-1 pathway in regards to human
chordomas, some interesting things were discovered through this experiment. PD-L1 expression seemed to be
localized on tumor infiltrating immune cells, rather than on the tumors themselves. This, along with the knowledge
that tumor cells have the ability to up regulate PD-L1 expression through pro-inflammatory cytokines, suggests that
the tumor cells were under minimal pressure by the immune system. In the circumstance of any immunologic
pressure, tumor cells would have been expected to express PD-L1 at higher levels in order to escape the immune
response. This experiment also showed that the media in which the cells were cultured affected the baseline and
inducible expression of PD-L1. JHC7 cells cultured in FBS containing media, as opposed to stem cell media,
expressed much lower levels of PD-L1. A further examination of the experimental results suggested that post-
transcriptional regulation of PD-L1 and PD-L2 must play a significant role in their activity. This inference was made
due to the observation that although PD-L1 and PD-L2 were not up regulated at the protein level in the U-CH2 cell
line, they were significantly up regulated at the mRNA level when stimulated with IL-4 (Mathios, D., et al. 2015).
Article 2: Introduction
PD-1 is a co-inhibitory molecule, present on T cell membranes, that is involved in the modulation of T cell
effector activity at sites of malignancies. When PD-1 is bound to it’s ligand, PD-L1, T cells become (or remain)
inactive. This effect of PD-L1 binding is thought to be a method by which tumors escape the body’s immune
response. Knowledge of this pathway is particularly useful when dealing with the limits of adoptive cellular
immunotherapy. Adoptive cellular immunotherapy is a promising method of cancer treatment in which active T
cells, against a specific targeted source, are gathered, expanded in vitro, then re-infused into the affected host
(Montes, M., et al. 2005). This therapy, however, has been limited by loss of function of the transferred T cells over
time. This loss of T cell function can likely be attributed to tolerisation by dendritic cells, or through direct
5. inactivation by the tumor itself due to the overexpression of PD-L1 on these cells. The most potent immunologic T
cell therapy lies within a form of T-memory cells known as Tcm cells. These cells are very effective, in that they can
mount a quick secondary immune response to a particular pathogen, but they express higher levels of PD-1 than do
naïve T cells. The following experiment seeks to prevent the loss of effector function of Tcm phenotype CD8+
T
cells through a blockade of the PD-1/PD-L1 pathway (Blake, S. J., et al. 2015).
Methods
Mice utilized for this experiment included three separate strains known as CD45.1+
OT-I, 11c.OVA and a
non-transgenic control strain. CD45.1+
OT-I mice were significant in that they expressed ovalbumin (OVA)
receptors on their T cells. The T cells of these mice were used to target OVA expressing tumor cells by adoptive
immunotherapy. The transgenic 11c.OVA breed was selected because the mice of this breed expressed OVA on
their self-cells (Steptoe RJ, et al. 2007). Naïve T cells were taken from the CD45.1+
OT-I mice and cultured in
media containing recombinant human IL-2 for three days (Blake, S. J., et al. 2015). IL-2 is a cytokine that stimulates
the differentiation of naïve T cells to effector and memory t cells (Gaffen, S. L. and K. D. Liu 2004). After three
days, these cells were harvested, washed, and then cultured for another 2 days in a media containing recombinant
mouse IL-15. IL-15 is another cytokine, which functions to promote the proliferation of CD8+
T cells (Mathieu, C.,
et al. 2015). This culture generated a large population of Tcm-like CD8+
T cells, which were then injected into both
the non-transgenic and the 11c.OVA mouse breeds. Some mice were instead injected with naïve T cells, to test
whether or not a PD-1/PD-L1 blockade would affect their blocked activation by tolerising dendritic cells.
Populations of monoclonal antibodies, specific to either PD-1 or PD-L1, were injected into mice every 3 days. These
antibodies, known as αPD-1 or αPD-L1 served to block the PD-1 receptors or the PD-L1 proteins. A line of cancer
cells known as B16.mOVA was prepared by transfecting B16-F0 cells from a mouse melanoma cell line with a
pcDNA3.1 encoding an ovalbumin fusion protein. The resulting cancerous cells then expressed OVA, which would
then cause them to be a target for the OT-I T cells. The B16m.OVA cells were injected into the two aforementioned
mouse breeds. The tumor sizes were measured daily and mice were euthanized when tumor areas exceeded 1cm2
.
Some mice injected with the cancer cells were not injected with the αPD-1 or αPD-L1 in order to serve as a control.
Presumably, the tumors of these mice should’ve grown faster than those of the mice injected with the monoclonal
antibodies. In order to determine CD8+
T cell effector activity in vivo, single cell suspensions of spleen cells were
prepared, as the spleen is a primary location for Tcm cells in the body
(Clemente, T., et al. 2013). Of these
6. suspensions, half were pulsed with OVA and fluorescently labeled. All spleen cells were then injected into the mice,
and three days later their spleens were collected and analyzed by flow cytometry to determine cytotoxic lymphocyte
activity by percentage of killing of OVA-pulsed targets in the test vs. the control. (Blake, S. J., et al. 2015).
Results
After injecting 11c.OVA mice with OT-I naïve T cells to test whether or not PD-1/PD-L1 blockade would
prevent dendritic cell mediated tolerance of naïve T cells, the results showed that after administration of αPD-1 or
αPD-L1, the magnitude of Tcm cell accumulation was far greater after three days than it was in mice that were not
given antibodies. Furthermore, the Tcm cell population persisted for a longer period of time in the mice that were
given antibodies. In mice that were not given either of the antibodies, T cells did proliferate substantially, but their
deletion followed along with only a small population of tolerant T cells. Following these results, an experimental
procedure was conducted to see if tolerance, of naïve T-cells could be reversed by a PD-1/PD-L1 blockade. Both
11c.OVA and non-transgenic mice were injected with naïve OT-I T cells. After a period of 21 days, when most cells
had been exhausted, αPD-1 or αPD-L1 was introduced. Half of each of these mice were also injected with
OVA/Quil A, an adjuvant containing ovalbumin, in which its purpose was to invoke a greater immune response. The
non-transgenic mice demonstrated a great (>80-fold) response in OT-I T cell proliferation to the stimulation with
OVA/Quil A before the antibodies were administered. The administration of αPD-L1 resulted in a 7-fold expansion
of OT-I T cells after the OVA/Quil administration in the non-transgenic mice. In these same mice, OT-I T cell
expansion did not increase significantly after administration of αPD-1. In the 11c.OVA mice, OT-I proliferation did
not increase as drastically either after stimulation by OVA/Quil A, or after administration of antibodies. To decipher
whether or not this expansion of OT-I T cells resulted in effector activity, levels of IFN-γ were measured, as IFN-γ
production results from T cell effector activity. The levels of IFN-γ increased over 1000-fold in the non-transgenic
mice, corresponding well with the increased OT-I T cell proliferation results. A similar method was used to
determine if the inactivation of OT-I Tcm cells could be reversed by a PD-1/PD-L1 blockade. The results of this
expressed that the blockade, along with OVA/Quil challenge, could drastically increase T cell expansion and
function in non-transgenic mice, but could not do so substantially in 11c.OVA mice. When it came to the 11c.OVA
mice that were infused with OT-I Tcm cells for adoptive immunotherapy, immediate treatment with αPD-L1 caused
persistence of effector function of CD8+
T cells, whereas cells treated with the isotype control did not exhibit this
effect. The in vivo T cell effector function was also verified, in that the percentage of OVA-pulsed spleen cells that
7. were killed was far greater after antibody administration in 11c.OVA mice. Finally, after graphing the percentage of
mice surviving vs. days post-inoculation, it was concluded that adoptive cellular immunotherapy, combined with the
blockade of the PD-1/PD-L1 pathway, led to a greatly increased survival time. In some cases, the survival time was
more than doubled by this treatment. Furthermore, this combined treatment led to decreased tumor sizes in most of
the mice (Blake, S. J., et al. 2015).
Discussion
Adoptive immunotherapy has been an effective, but limited, treatment for many cancers. The results of this
experiment showed that this type of therapy can become more effective in a tolerogenic environment if combined
with a blockade of the PD-1/PD-L1 pathway. When comparing the results of this experiment with the results of
similar experiments, the anti-tumor affect of PD-1/PD-L1 blockade in combination with a secondary treatment type,
such as vaccination or blockades of other co-inhibitory pathways, is further established. When all of the results of
this experiment are combined, they show that a PD-L1 blockade promotes adoptive immunotherapy by increasing
the proliferation and conversion of CD8+
T cells to cytotoxic T lymphocytes before deactivation by dendritic cells.
They also demonstrate that, as a result of this cytotoxic T lymphocyte accumulation, there is an increased infiltration
of these cells at the tumor site, and a decreased frequency of T cell exhaustion. Together, the results conclude that
co-inhibitory pathway blockades may be a promising route for cancer treatment (Blake, S. J., et al. 2015).
Synthesis/Follow-up
The two reviewed articles delved into and summarized some of the most prominent features of the PD-
1/PD-L1 co-inhibitory pathway in regard to cancer cases. Research of the PD-1/PD-L1 pathway is extremely
significant when it comes to progressive cancer treatments, as it provides hope for a more natural therapy for many,
if not all types, of aggressive cancer. This type of treatment, involving reduction of the suppression of ones own
immune system, would potentially be a safer and more effective form of therapy than ones that are currently widely
used, such as chemotherapy.
Between article one, which showed that PD-1 and PD-L1 expression could be up regulated in chordomas
by the cytokine IFN-γ, and article two, which showed that a blockade of PD-1/PD-L1 promoted increasingly
successful adoptive immunotherapy, some interesting correlations can be seen. For example, in the second article, it
was noted that IFN-γ was substantially increased with increased effector activity. Based on the results of article 1,
this seems to confirm that tumor cells and dendritic cells can up-regulate their PD-L1 expression in the vicinity of
8. activated cytotoxic T cells. This is important to note, as IFN-γ or other pro-inflammatory cytokines, which IFN-γ
leads to the production of, may serve as somewhat of a warning signal to tumor cells that they are under attack. With
this knowledge, it may be worthwhile to investigate a method with which to locally de-sensitize tumor cells to IFN-
γ. Recognition and response to IFN-γ by cells has been shown to be heavily dependent upon the β chain of the IFN-γ
receptor (Bach, E. A., et al.1997). Since tumors seem to have the ability to respond to this cytokine, it can be
inferred that they possess an active β chain. A possible technique to down-regulate a tumor’s response to IFN-γ
would be to produce antibodies against this β chain. If this experiment proved to be successful, it would likely
reduce tumor cells’ expression of PD-L1 in the presence of effector T cells, and thus reduce the need for an antibody
blockade of PD-L1 or PD-1, successfully lowering the risk of an autoimmune attack. Autoimmune responses are a
particular concern for this area of research, as PD-L1 expression by healthy cells serves as a method of preventing
their attack by T lymphocytes (Role of PD-L1 Pathway, 2015).
Another intriguing element, derived from article 2, was not deeply discussed by the authors. It is very
peculiar that the T cell effector activity was exponentially increased in non-transgenic mice by stimulation with
OVA/Quil A, even before the αPD-1 or αPD-L1 monoclonal antibodies were introduced. In this situation, cytotoxic
T cells proliferated and responded, even in the presence of PD-L1 expression. This was due to the fact that the OT-I
T cells had receptors specific to OVA and were able to elicit a quick immune response, which might also lead one to
the conclusion that other components, such as recognized antigen presentation to T cells, could override the PD-
1/PD-L1 pathway. Non-cancerous cells with the ability to undergo apoptosis, initiated by T lymphocytes, when
presenting pathogenic or damage associated peptides strengthens this hypothesis. If this hypothesis were correct,
then introducing a familiar pathogen, perhaps a virus, deep within the tumor should produce a T cell response that
would overlook the PD-L1 expression. In this experiment, it would be a better choice to choose a pathogen which
the person has either fought off in his or her life, or been vaccinated against. The reason for this is because, as
discussed in article 2, Tcm cells have the greatest and most efficient potential therapeutic effect. The introduction of
a familiar pathogen into the tumor environment would most likely elicit a B memory cell response as well. B cells,
during a secondary immune response, are able to quickly recognize antigens and differentiate into plasma cells,
which produce antibodies specific to the foreign molecules. These antibodies could then opsonize the tumor cell and
promote phagocytosis by macrophages along with increased cytotoxic T cell activity. This method of tumor
9. treatment, if effective, would essentially act as a post-exposure vaccine, and would not require the blockade of PD-
L1 or PD-1 ideally.
A final item of interest came about when reading through article 1. As discussed previously, IFN-γ and IL-
4 are cytokines with antagonistic properties to one another. Levels of expression of PD-1 and PD-L1 were measured
at base level and after one exposure. In most cases, IFN-γ caused the levels of expression to rise, especially the
levels of PD-L1. However, IL-4 didn’t have a large effect on the base level of expression. One ought to further this
experiment by testing the effect of IL-4 on elevated levels of PD-1 or PD-L1 expression. Would IL-4 actually serve
to lower the expressions of these proteins? If so, the effects of IL-4 and IFN-γ in combination should be tested on
cells expressing basal level and cells expressing high levels of the proteins. If IL-4 has the capability of lowering
PD-L1 or PD-1 expression, then it may be a viable treatment option for certain tumor environments expressing high
levels of PD-L1.
The PD-1/PD-L1 pathway is a relatively recently discovered co-inhibitory pathway that shows great
promise for the treatment of cancer. It is important, when researching this pathway, to avoid possible autoimmune
responses, as healthy self-cells use this pathway as a means of avoiding attack. Most self-cells, however, express
PD-L2 as well as PD-L1, where some tumor cells only express PD-L1 (Role of PD-L1 Pathway, 2015). Therefore,
PD-L1 seems to be the most effective target when dealing with blockade of the pathway by monoclonal antibodies.
There may be other successful ways in which the body’s immune system could dodge this pathway when targeting
tumor cells, as discussed. These methods, however, have yet to be studied, and there are limits to which types of
cancers can be studied in mouse models, because effective mouse models for certain cancers such as chordomas are
not yet available.
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doi: 10.4049/jimmunol.178.4.2094
