Clinical and Applied Immunology Reviews 5 (2005) 149–166

Tumor infiltrating lymphocytes (TILs): Lessons learned

in 30 years of study
Kristen M. Dreschera,*, Henry T. Lynchb
a
Department of Medical Microbiology and Immunology, Creighton University School of Medicine,
Omaha, NE 68178, USA
b
Department of Preventative Medicine; Creighton University School of Medicine, Omaha, NE 68178, USA
Received 9 September 2004; received in revised form 18 January 2005; accepted 25 March 2005.

Abstract
The interplay between tumor development and the host immune system, as well as the impact of
the immune system on the tumor’s metastatic potential is incompletely defined. Almost 30 years
ago, the identification of tumor infiltrating lymphocytes was reported, and represented great hope in
cancer treatment. At that time, it was thought that patients whose tumors had high numbers of tumor
infiltrating lymphocytes had a good prognosis, while patients with few or no tumor infiltrating
lymphocytes had a poor prognosis. Work published since that seminal report has indicated that this
viewpoint is overly simplistic. While infiltration of the tumor with lymphocytes may be one factor
associated with a positive outcome, the milieu required for optimal functioning of the immune system
is also defined by the presence of potent antigen presenting cells, immunostimulatory cytokines, and
optimal surface molecule expression by both the tumor cells and the infiltrating lymphocytes. The
complexities of these interactions are just beginning to be defined; a further difficulty that must
be addressed in the development of cancer immunotherapy regimens is that various forms of cancer
appear to have different immune system requirements. Failure of the host to achieve the optimal
tumor microenvironment severely compromises the ability of the host to control tumor growth and
metastases. Animal models of cancer, imperfect as they are, can provide investigators with model
systems for manipulating the immune parameters of the tumor site within the host to determine its
effect on disease outcome. While mouse models cannot fully mimic the processes observed in
humans because of differences in surface molecule expression and signaling pathways between the
species, they do provide investigators with a means to examine the mechanisms involved following

Abbreviations: APC, antigen presenting cell; CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T lymphocyte
antigen-4; FasL, Fas ligand; ICAM, intercellular adhesion molecule; IEL, intraepithelial lymphocyte; IFN,
interferon; IL, interleukin; LFA, lymphocyte functional antigen; MHC, major histocompatibility complex; MSI,
microsatellite instable; MSS, microsatellite stable; NK, natural killer; TILs, tumor infiltrating lymphocytes;
T regs, regulatory T cells; TCR, T cell receptor; TGF-β, transforming growth factor-β; TH, T helper; VLA, very
late antigen.
* Corresponding author. Tel.: ⫹1 402 280 2725; fax: ⫹1 402 280 2875.
E-mail address: kdresche@creighton.edu (K.M. Drescher)

1529-1049/05/$ – see front matter 쑕 2005 Elsevier Inc. All rights reserved.
doi: 10.1016/j.cair.2005.03.002
150 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166

immunotherapy. This review will discuss some of the immune parameters studied in tumor
infiltrating lymphocytes and how they have been associated with patient prognosis.
쑕 2005 Elsevier Inc. All rights reserved.
Keywords: TILs; Cancer; Cytokines; Fas; Adhesion molecules

1. Introduction
Cancer is responsible for more than 500 000 deaths in the United States per year, and ranks
second only to heart disease as the cause of death in Americans [1]. While death rates due
to heart disease have dropped precipitously since 1950, deaths rates due to cancer have remained
relatively steady. The standard approach to treating most forms of cancer consists of surgery
followed by radiation and/or chemotherapy; nonetheless, cancer often metastasizes widely, that
is, patients with colorectal cancer frequently develop liver cancer. While approximately 25%
of colorectal cancer patients have liver metastases at diagnosis, an additional quarter of the
remaining patients develop liver metastases within 2 years of diagnosis [2]. Why some patients
are refractory to developing metastases is unknown, but one hypothesis is that the immune
system of some individuals is more effective in controlling metastatic events than that of
other patients. In considering the factors that would contribute to an optimal immune response
against tumors, one would assume that the same factors critical to an immune response to any
antigen would be similar to those involved in the body’s ability to control tumors; these
include antigenicity of the tumor, ability to recruit an optimal mixture of cell types to
the tumor site, suitable cytokine production by both the tumor and the infiltrating cells, and
expression of cell surface molecules that enhance immune responses. In addition to these
factors we are now beginning to realize that the tumor, like many bacteria and viruses, has
the ability to alter the microenvironment to promote survival and dispersement of the tumor
cells or conversely, its failure to mount such an immune response may result in death.
The concept that the adaptive immune system has an intrinsic ability to control tumor
growth and spread via the adaptive immune response originated over 50 years ago with a
series of tumor transplant experiments in mice [3]. In these studies, carcinogen-induced
tumors were resected from animals and then transplanted into either the same mouse or a
syngeneic mouse; the mouse that the tumor was originally obtained from resisted tumor
growth, while the naive syngeneic mouse was unable to prevent tumor growth [3]. These
studies clearly indicated that the adaptive immune system can be stimulated to respond to
a particular tumor, and that this response is sufficient to reject subsequent challenges with an
identical tumor. Despite the promise of these early data, in vivo the human immune system
does not respond to tumors in this manner. Of course, there may be significant numbers of
cancers that never develop because of efficient antitumor responses, but this cannot be
quantified. The rare phenomenon of spontaneous regression may also be the result of success-
ful immune control. The choice of tumor in these early studies (methylcholanthrene-induced
sarcomas) may have been fortuitous because many tumors are weakly antigenic and express
modified host proteins on their surface. Conventional wisdom would suggest that when
tumors express unique antigens on their surface, the immune system could be stimulated to
control their growth and metastases. Our knowledge to date suggests that if such responses
 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166 151

occur in vivo, other mechanisms can be invoked to actively suppress the desired antitumor
responses in many instances. Gene polymorphisms, particularly in genes associated with the
immune response (especially cytokine genes), have also been associated with host prognosis
in different forms of cancer [4–8].
The inherent nature of many tumor antigens makes it difficult for the host to mount a
strong antitumor response. Tumor antigens are often aberrantly expressed host proteins that
are the result of developmental dysregulations or small alterations in the normal protein. As
in an immune response to any antigen, these proteins must be present at levels sufficient to
stimulate an immune response when presented to T lymphocytes by major histocompatibility
complex (MHC) molecules on the surface of an antigen presenting cell (APC). Because
tumor antigens are often derived from proteins that are part of the normal host makeup,
T cells that are normally responsive to peptides derived from these proteins may be deleted
during thymic selection, or peripheral tolerance is induced if the T cell’s first encounter with
peptide is on the surface of an APC that does not express the correct costimulatory molecules
on its surface. A naive T cell requires interaction with professional APCs (such as dendritic
cells or macrophages) for these initial encounters to prevent anergy of the T cell from
occurring. Proteins unique to immune privileged sites (ie, retina, brain, and testis) are normally
sequestered from the immune system; there are naive T cells in the periphery capable of
responding to these antigens if they are expressed on the tumor. Examples of these types
of antigens include MAGE-1 and MAGE-3 that are expressed in some forms of breast and
brain cancers [9,10]. These proteins are normally expressed in the testis. Because the
testis is inaccessible to the immune system under normal conditions, T cells reactive to
peptides derived from this protein are not anergized and are found in the periphery of the host.
Clinical examples that suggest that there is a subset of tumors with a strong degree of
antigenicity abound. Thirty years ago, Murray et al. [11] studied 148 patients and found that
individuals with high levels of inflammatory cells in their tumors had a better prognosis
than those without tumor infiltrates. Since this report, numerous immunohistochemical
studies have been performed on a wide variety of tumor types (ie, uterine cervical [12],
colorectal [13,14], esophageal [15], breast [16], ovarian [17], renal [18]) to characterize the
tumor infiltrating lymphocytes (TILs) and correlate the data with patient prognosis. The ratio-
nale for these studies was that insight into tumor control mechanisms used by the host
would be provided; by exploiting these naturally occurring control mechanisms, effective
immunotherapies could be developed. Unfortunately, the reality of the data from these studies
was less clear-cut than that anticipated in the early work. While patients with increased
numbers of TILs often have increased survival rates, this is not always the case [13,19–24].
A variety of explanations have been offered for these findings including the nature of
the infiltrate, the antigenicity of the tumor, ineffectiveness of antigen presentation, and the
soluble mediators expressed within the tumor microenvironment.
The underlying etiology of a particular cancer would a priori be expected to influence
the tumor infiltrate, given the diverse origins of human cancers, which suggests that the host
may have multiple strategies to control or alternatively, fail to control, malignancies. It is
therefore not surprising that there is no consensus regarding the type of cells or their
products that are required for optimal tumor control. In the case of cervical cancer, in which
a large percentage of the cancers are attributed to infection with human papilloma virus [25],
152 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166

an effective immune response may be inherently different than cancers caused by a nonviral
etiologic agent, such as radiation exposure. While cytotoxic T lymphocytes are presumed
to be major participants in killing cancer cells as well as responding to viral infections, it
is difficult to predict what other cell types and immune products are important for an effective
defense in cancers attributed to multiple genetic events, which may or may not be further
influenced by various pathogenic environmental agents.
There is no agreement regarding the utility of examining human tumor specimens for
infiltration by lymphocytes as either a prognostic indicator or a means to guide treatment
options for the patient. Individuals with carcinomas bearing particular characteristics, such
as colorectal cancers with high levels of DNA microsatellite instability, have increased levels
of TILs and a better prognosis than patients with low levels of DNA microsatellite instability.
In addition, colorectal cancer patients with high levels of DNA microsatellite instability re-
spond differently to chemotherapy [26,27]. If these 2 traits, level of microsatellite instability
and TILs, were highly correlated with each other as well as a particular prognosis, physicians
could make targeted treatment decisions based on an examination of a simple pathology
sample rather than on requiring more technically demanding and expensive molecular
biology testing, which may not be available to some health care providers.
The main mechanism by which TILs control tumor growth is postulated to be via a
cytotoxic mechanism; TILs may also produce cytokines such as interferon-γ that potentiates
a cellular immune response. In this scenario, infiltration of the tumor site with high numbers of
CD8⫹ TILs would be desirable. CD4⫹ TILs would also be required because CD8⫹ T cells
usually need CD4⫹ T cells to function optimally. The ratio of CD4⫹ to CD8⫹ T cells is likely
a key to appropriate TIL function; this ratio may be different for different forms of cancer.
An examination of cervical carcinoma samples found that patients with lymph node metastases
had lower levels of CD4⫹ T cells within the tumor than those without metastases, supporting
a role for CD4⫹ T cells in tumor control [12], and illustrating the critical balance of cell
phenotypes needed to prevent the tumor escaping from the control of the host immune re-
sponse [12]. One of the key functions of CD4⫹ T cells is likely related to their soluble
mediator (cytokine) production; these cytokines provide CD8⫹ T cells with growth factors
required for expansion during an immune response. In this review, we will discuss some of
the parameters involved in mounting an efficient antitumor response. We will discuss the
characteristics of both the APC and the infiltrating lymphocytes that can impact tumor control,
as well as outline some of the tumor characteristics that may have a negative impact on
the host immune system. Fig. 1 details some of the factors that we will cover in this review.

2. Dendritic cells
Dendritic cells are the most potent APCs in the body. These cells are efficient at priming
naive T cells because they express high levels of costimulatory molecules such as B7.1
(CD80), B7.2 (CD86), and CD40 on their surface. In the absence of high levels of costimula-
tory molecules on an APC, a naive T cell becomes anergized upon its first encounter with
antigen, thus making the host unresponsive to that particular antigen for life. Dendritic cells also
have the capacity for cross-priming, a process involving presentation of exogenous antigens
 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166 153

Fig. 1. Interactions between the tumor cell, T cell, and APC (in this example, a dendritic cell) within the host.
This diagram summarizes some of the key interactions that occur during an antitumor response. Green, red, and
blue letterings indicate that the protein is produced by the T cell, the tumor cell, and the dendritic cell, respectively.
Items in a bracket [], indicate that the proteins have a negative impact on the host immune response to the tumor
when they are produced. Items not bracketed are generally desired for an optimal antitumor response.

to CD8⫹ T cells, which may be critical in triggering an immune response to some tumor
antigens, because tumors often express low levels of MHC Class I on their surface. Several
studies have reported that patients with large numbers of infiltrating dendritic cells within
their tumors have a better prognosis than those with low levels of dendritic cell infiltration [28–
32]. Additionally, patients with limited numbers of dendritic cells infiltrating into the tumor
site also had an increased incidence of metastases [31].
Reichert et al. [28] performed immunostaining on samples from patients with primary
oral squamous cell carcinoma to determine if there was a correlation between the number of
dendritic cells and prognosis. Using S-100 as a marker of dendritic cells, a significant
correlation was found between low dendritic cell density and poor patient survival. An
interesting observation was that TILs within tumors infiltrated with low levels of dendritic
cells had abnormal expression of the ζ chain of the CD3 complex on their surface [28].
Because ζ chain is the component of the CD3 complex responsible for transmitting the
signal to the T cell once ligation of the T cell receptor (TCR) occurs, these TILs cannot
154 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166

respond appropriately to antigen. Because of this association between the loss of the ζ chain
and the low levels of dendritic cells, one could reasonably hypothesize that the immunosuppres-
sion observed in certain forms of cancer may be due to the interactions between APCs and
T cells, and not solely a function of TIL density. A similar correlation between ζ chain
expression and lymph node metastases has also been noted in a study of patients with gastric
cancer [33].
The study of human dendritic cells in colon cancers found that the density of dendritic
cells in cancers that had metastasized was 6-fold lower than that in primary colon cancers;
in addition, dendritic cell density in primary colon cancer samples was lower than that in
the normal colon [34]. This finding suggests that factors within the tumor microenvironment
limit either the infiltration or development of the dendritic cells. Despite the immunosuppres-
sive nature of some cytokines such as transforming growth factor (TGF)-β and interleukin
(IL)-10, no significant correlation was found between expression of these mediators and the
level of dendritic cell infiltration; however, increases in tumor necrosis factor α were associ-
ated with increased dendritic cell infiltration [34]. This association between cytokine expres-
sion and dendritic cell infiltration represents a subtle change in viewpoint from conventional
thought—instead of the tumor products actively suppressing infiltration of immune cells, it
may be more fruitful in some instances to examine alterations in proinflammatory mediators.
A similar attempt to correlate dendritic cell numbers with survival in patients diagnosed
with pancreatic adenocarcinomas was unsuccessful because of the scarcity of both dendritic
cells and lymphocytes [35].
Because of their ability to take up, process, and present antigen, significant efforts are being
focused on developing immunotherapy approaches that enhance the function of dendritic cells
[36,37], with the hypothesis that enhanced dendritic cell function will result in increased
cytotoxic T lymphocyte generation, thereby allowing the host to control tumor growth. The
influence of dendritic cells on tumor control and metastases is complicated by the existence of
various dendritic cell subsets derived from blood monocytes, hemopoietic precursors, or
skin [38]. Many studies have focused on the use of dendritic cells pulsed with antigen as a
means of cancer immunotherapy [39–41]. While these types of studies have been successful
in small animal models, the success and practicality of this approach in clinical medicine
remains to be seen.

3. CD4ⴙCD25ⴙ T regulatory cells: a balance between tumor responses

and autoimmunity?
Regulatory T cells (T regs) are a population of CD4⫹CD25⫹ T cells that participate in
immune regulation via the suppression of immune responses. This subpopulation of CD4⫹
T cells is a key in maintaining self-tolerance in the host; their major function appears to be
preventing the development of autoimmune diseases. These CD4⫹ T cells also produce high
levels of IL-10. In several animal models of autoimmunity, the transcription factor Foxp3
has also been designated as a marker of T regs [42]. Regulatory T cells are global suppressors
of immune function, impacting both CD8⫹ T cells and CD4⫹CD25⫺ T cells. In murine
models of autoimmune disease, in vivo depletion of CD4⫹CD25⫹ T cells increases the level
 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166 155

of organ-specific autoimmunity [43]. While it is accepted that T regs are critical in maintaining
self-tolerance, one must consider the price the host may pay as a consequence of possessing this
immunosuppressive cell population. For example, is the host more susceptible to certain
infectious agents or some cancers due to the presence of this CD4⫹CD25⫹ population of
T cells?
While TILs are described in many forms of cancer, high levels of TILs are rarely associated
with a complete cure. One could argue that a partial explanation of this observation is
that cancer patients are frequently immunosuppressed due to the treatment regimens used.
However, cancer patients with normal levels of peripheral lymphocytes often have poor
recall responses to antigens in vitro. Experimental paradigms using mouse models of
cancer have suggested that the immunosuppression may be due to an expansion of T regs.
In vivo depletion of CD25⫹ cells via the administration of an anti-CD25 antibody restored
antitumor responses in animals formerly unresponsive to the tumor [44]. The animals also
experienced an increased responsiveness to antigens that were not expressed by the tumors,
demonstrating that these cells impact an array of responses in the host. In a murine model
of progressive breast cancer T regs were detected within 14 days of tumor challenge, and
this population persisted throughout the course of the experiment [44], supporting the
view that there is an intrinsic mechanism that operates to suppress the immune responses. It
is significant that these cell types appear early in the immune response to tumors and
consequently, suggests that tumors devise immune evasion strategies almost immediately
upon development.
Recent work in humans has examined whether patients with cancer have altered levels
of T regs in the periphery compared with healthy individuals, or if there is an association
between the level of T regs and cancer stage. Ichihara et al. [45] reported a significant increase
in the number of T regulatory cells in the periphery of patients with either gastric or esophageal
cancer compared to control subjects. The CD4⫹CD25⫹ cells isolated from gastric cancer
patients secreted IL-10 but not interferon-γ and suppressed proliferation of autologous
CD4⫹CD25⫺ T cells, supporting the role of these cells as immune response inhibitors. In
another study, CD4⫹CD25⫹ T cells from cancer patients also expressed cytotoxic T lympho-
cyte antigen-4 (CTLA-4) and CD45RO on their surface. The regulatory cells in the periphery
of this set of cancer patients also produced TGF-β [46]. Cytotoxic T lymphocyte antigen-4
is a surface molecule associated with inhibition of T cell activation and is also a marker
associated with T regs. Another study found increased levels of CD4⫹CD25⫹ TILs in
tissue samples from gastric cancer patients compared to those of CD4⫹CD25⫹ normal
intraepithelial lymphocytes in the normal gastric mucosa from control subjects [45].
It is important to note that not all CD4⫹ T cells expressing CD25 on their surface are
classified as T regulatory cells. Other molecules such as CTLA-4, foxp3, and GITR are also
expressed in this subset of T cells, at least in mice. The mechanism of the suppression elicited
by T regs is incompletely defined, but some mouse studies suggest that this immune
suppression cannot be solely attributed to the cytokines produced by the T regs and that
cell-cell contact is required [47].
In considering the potential for immunotherapy in the treatment of cancer one must consider
the potential hazards involved in immunizing against tumor antigens. Could immunotherapy
potentially do more harm than good without a complete understanding of the factors that
156 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166

control T reg development? If one can overcome these hurdles and sufficiently suppress
T reg development to stimulate the antitumor response what are the long-term consequences
to the host? Experimental studies in mice suggest that the balance between controlling
autoimmune responses and enhancing immune responses to tumors is a delicate one, and
disrupting and altering this balance may predispose the host to autoimmune disease because
immunotherapy aimed at increased antitumor immunity [48,49]. Could those treated for cancer
develop equally devastating multiorgan autoimmune diseases because of immunotherapy? An
understanding of the development, functions, and control of T regs remains critical to our
ability to treat and control tumors.

4. Fas-mediated counterattack: a mechanism of immune privilege exploited

by tumors
Killing of target cells by natural killer or CD8⫹ T cells occurs via the perforin/granzyme
pathway or the Fas/Fas ligand (FasL) pathway. The end result of both pathways is apoptosis
of the target cell. Fas ligand is a member of the tumor necrosis factor family; ligation of
FasL transmits a death signal to the Fas (CD95/APO-1)-expressing target cell. T lymphocytes
can express both Fas and FasL on their surface. While it is obvious that it would be desirable
for cytotoxic T cells to express FasL on their surface, Fas expression by the T cell has 2
potential roles: first, ligation of Fas on the T cell induces apoptosis of the cell, which resolves
an immune response once an antigen has been cleared from the host; second, Fas expression
by the T cell may be involved in maintaining a state of immune privilege in some tissues,
although this is a matter of considerable debate [50,51]. Some correlative studies with human
tissue suggest that some cancers have exploited this second mechanism to subvert the immune
response and escape from immune surveillance [14,15,24,52–58].
Fas-mediated killing of tumor cells occurs when a Fas-positive T cell delivers a signal
to a Fas-bearing tumor cell and the tumor cell undergoes apoptosis. Despite infiltration of the
tumor site with an optimal ratio of CD4⫹/CD8⫹ T cells with the correct TCR, TILs may
not control tumor growth and/or metastases in this scenario. Several studies using human
tissue have correlated Fas and FasL expression with tumor cell death [13–15,23,24,59–61].
Expression of FasL on the TIL and Fas on the tumor cell would permit the TIL to deliver
a death signal to the tumor cell, resulting in apoptosis of the Fas-bearing tumor cell. Expression
of membrane-bound FasL on the tumor cell would lead to the opposite effect, deletion of
the TIL. This process of immune evasion has been termed Fas counterattack [15] and
described in humans in cancers of the colon [14,52], esophagus [15], skin [53], stomach
[54,61], liver [13,58], and breast [60], as well as in angiosarcomas [14,24,57]. Many studies
correlating TIL density or characteristics with prognosis have shown that TIL density corre-
lates with enhanced survival [19–22]. In cases where there are exceptions to this, frequently
high levels of apoptotic TILs have been noted, suggesting that tumor infiltration by lympho-
cytes is not a sufficient prognostic indicator of patient survival; rather, one must also consider
the level of apoptotic TILs as well as FasL expression by the tumor cells to accurately
predict survival [15,53,55,61].
A recent study by Asanuma et al. [62] has provided insight into a potential mechanism
as to how FasL-mediated immunosuppression is induced. The role of survivin, a member
 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166 157

of the inhibitor-of-apoptosis family, in Fas counterattack was examined. Immunostaining

experiments using human colon cancer tissue demonstrated a correlation between staining
intensity for survivin and FasL. Transfection of LS180 cells with a plasmid encoding
survivin resulted in increased FasL expression on the cells’ surface. Subsequent transfection
of these cells with survivin-specific small inhibitory RNA downregulated FasL expression
suggesting that survivin is a key contributor to FasL induction by tumor cells. While it is
premature to predict whether survivin-mediated upregulation of FasL will represent a general
mechanism used by a broad array of cancers to control the host immune response, it does
potentially provide investigators with a unique therapeutic target for further study.
Whether FasL always induces a state of immune privilege in a tumor is controversial.
Houston et al. [63] suggested that perhaps FasL expression on tumors played a dual role—
sometimes it was immunosuppressive, while under other conditions it would induce inflam-
mation—thus enhancing the immune response. Based on work done in the field of allograft
rejection the authors hypothesized that in the absence of TGF-β1 FasL expression triggered
neutrophil recruitment, which would be detrimental to tumor growth and/or survival [64].
No correlation between FasL expression in colon cancer, neutrophil recruitment, and TGF-β1
expression was found in tissues from human colon cancer patients, although FasL was
correlated with increased levels of TIL apoptosis [63].
In a study of colorectal cancer patients [52], tumor samples were evaluated for TIL density,
DNA microsatellite instability level, FasL expression, tumor type, and the level of apoptotic
TILs. The authors reported that apoptotic TILs most often localized to tumors classified as
microsatellite stable. Additionally, a correlation between the number of apoptotic TILs and
tumor progression and metastasis was uncovered. Contradictory to the authors’ initial hypoth-
esis that high levels of FasL would be localized to tumors with the fewest TILs, this study
reported that tumors with the highest FasL levels were categorized as MSI-High; MSI-High
cancers were those with a more favorable prognosis [52]. Perhaps one of the most important
findings to come out of this study was that microsatellite stable tumors had the highest level
of apoptotic TILs but not the high levels of membrane-bound FasL, implying that another
mechanism that does not involve membrane-bound FasL is involved in TIL apoptosis. An
alternative mechanism could also invoke processes that protect TILs from apoptotic cell
death. Other studies have examined FasL expression in both primary and metastatic
colorectal cancers and found increased FasL expression in metastatic vs primary carcinomas
[14,15,23], suggesting that FasL expression by the tumors may serve as a useful prognostic
indicator in some patients.
Other molecules involved in the apoptotic pathways include c-Myb and Bcl-x. The relation-
ship of these proteins to prognosis in patients with colorectal cancer has been examined
[65]. Immunohistochemical staining demonstrated increased levels of Bcl-x and c-Myb in
patients with a poor prognosis. To further understand the relationship between these proteins
and why they may be associated with a poor prognosis, LoVo cells, a human colon cancer
cell line, were transfected with complementary DNA encoding c-myb. Transfected cells
experienced a 2- to 3-fold increase in Bcl-x protein compared to control cells; increased
messenger RNA levels were also observed. Immunodeficient mice were then injected with
the c-myb overexpressing cells. Tumors from mice injected with the c-Myb–transfected cells
were 2.5 times larger than tumors from mice injected with control-transfected cells. Further,
158 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166

no apoptosis was detected in tumors from mice injected with c-myb–transfected cells [65].
Together, these data demonstrate that tumor cells have developed multiple mechanisms for
resisting death, and that immunotherapies aimed at merely targeting Fas-FasL interactions
may not overcome resistance of the tumor cells to apoptosis.

5. Adhesion molecules
The significance of cellular adhesion molecule expression as a prognostic indicator in
cancer is less well defined than many of the other factors discussed thus far. The arguments
detailing the pros and cons of adhesion molecule expression within the tumor site are
intriguing. Lymphocyte functional antigen-1 (LFA-1) on the surface of lymphocytes interacts
with intracellular adhesion molecule-1 or -2 (ICAM-1, -2) on the APC. This interaction
provides stability between the APC and the responding T cell, allowing appropriate signals
to be delivered from the APC to the T cell via the TCR. It is hypothesized that this interaction
between ICAM and LFA-1 is the initial interaction that occurs between the APC and T cell,
and this binding assists the TCR and peptide-MHC complex to find each other. In the absence
of these interactions, effective T cell signaling cannot occur.
Early work on human melanomas correlated increased adhesion molecule expression with
an increased risk of metastases in the patients [66]. While the rationale for this finding is
not intuitive, the authors offered an intriguing hypothesis. They proposed that ligation between
ICAM-1 positive tumor cells and LFA-1 positive T cells allowed the tumor cell to disseminate
to other sites in the body via the adhesion to the surface of a T cell [66]. Using immunohisto-
chemistry, more recent work has shown that ICAM-1 levels are lower in colon cancer patients
with metastases compared to patients without metastases; patients with ICAM-1 negative
tumors had a significantly worse prognosis compared with patients whose tumors were ICAM-
positive [67]. Alternatively, other studies on cancer patients have found increased ICAM-1
expression in metastatic melanoma lesions [47,68], as well as an association between increased
tumor size and metastases in gastric cancer [69]. One could easily envision a scenario where
increased ICAM-1 expression could enhance tumor control, given its role in stabilizing
the interactions between the cognate T cell and the APC. In the absence of adequate ICAM-1
expression, an appropriate signal would not be generated through the TCR. These high levels
of ICAM-1 expression may also come with a risk. While increased adhesion to T cells would
enhance antigen presentation, this increased adhesion to the T cells may also allow tumor cells
to disperse to other sites in the host, as proposed by Johnson et al. [66]. The differential effects of
high or low ICAM-1 expression may be related to the types of immune responses most critical
to controlling a certain type of tumor. If high ICAM-1 expression were found to be a
general mechanism of metastases, this indeed would provide a significant challenge to treatment.
Blocking therapies aimed at these molecules, while perhaps reducing metastatic events, would
also result in impaired lymphocyte function and recruitment.
Kitayama et al. [70] examined expression of several β1 and β2 integrins on TILs from
patients with colorectal cancer using flow cytometry. β1 Integrins include very late antigens
1-6, which bind to vascular cell adhesion molecule thereby allowing lymphocyte infiltration;
LFA-1 is a β2 integrin. Analysis of these patient samples revealed that CD8⫹ cytotoxic
 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166 159

TILs had decreased LFA-1 on their surface compared to the levels detected on peripheral
blood lymphocytes obtained from the same patients. In contrast, LFA-1 levels on CD4⫹
TILs and peripheral blood lymphocytes (PBLs) were similar, β1 integrin staining was reduced
on both CD4⫹ and CD8⫹ TILs, compared to the levels observed in the peripheral
blood lymphocytes. Functionally, the CD8⫹ TILs exhibited decreased adherence to both
ICAM-1 and vascular cell adhesion molecule-1–coated substrates compared with PBLs. In
addition, adherence to HT29 cells (a colon cancer cell line) was also markedly reduced [70].
The inability of the TILs to interact with the tumor cells demonstrated a mechanism as to
why the TILs failed to lyse the tumors. Without the ability to make appropriate contact
between the APC (whether it be on the dendritic cell presenting exogenous antigen or the
tumor cell presenting self-antigen) and the responding T cell, it is unlikely that tumor lysis
will occur, giving the tumor a distinct survival advantage in the host.
Patients with diffuse B-cell large-cell lymphoma and increased levels of TILs experienced
fewer relapses and better survival compared to patients with low TIL levels [71]. Correlated with
this finding was the observation that patients with diffuse large-cell lymphoma with low TIL
levels also had reduced expression of MHC Class I and Class II within the tumor. Further
examination of these patients for HLA, ICAM, LFA-1, ICAM-1, B7.1, and B7.2 expression
followed by categorizing the patients into subgroups based on TIL levels determined that
all patients with low levels of TILs were deficient in expression of at least one of the surface
molecules under study [72]. These data also indicate that minimally, one portion of the antigen
recognition and presentation process was lacking. While these data are not in agreement
with some other studies on diffuse large-cell lymphoma [73,74], the differences may be
attributed to the criteria used by the authors to analyze their samples.
The collective data in the field of adhesion molecule expression and function are contradic-
tory and difficult to sort out. The same molecules (such as ICAM-1) required for effective
immune function have been associated with metastatic events. These data challenge the idea
of one-size-fits-all treatment. Adhesion molecule expression may be a marker that would
benefit from monitoring throughout the course of treatment and adjustments to therapy can
be made accordingly.

6. Cytokines
The microenvironment, particularly the soluble mediators expressed, is known to impact
immune cell function. Likewise, the local cytokine milieu in the tumor is also correlated with
the immune response to the tumor. Transforming growth factor-β (TGF-β) is a multifunctional
mediator critical to the development of B, T, and natural killer cells; it is also involved in
the maintenance of immune privilege. Because of its role as an immunosuppressive agent,
serum levels of TGF-βl have been examined in cancer patients. In a study of patients suffering
from hepatocellular carcinoma, there was an inverse relationship between serum levels of
TGF-βl and tumor size. Patients with high levels of Fas on their CD4⫹ peripheral T cells
had lower levels of TGF-βl [75]. Consistent with these data, in vitro studies have shown
that treatment with TGF-βl decreased Fas expression and susceptibility to apoptosis in both
human dendritic cell precursors [76] and murine bone marrow progenitor cells [77]. Together,
160 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166

these data suggest that one potential mechanism for tumor evasion of the immune response
involves downregulating TGF-βl production, which upregulates Fas on the surface of TILs,
thus increasing their susceptibility to apoptosis.
The concept of T helper (TH) 1 and TH2 cells has also been explored in cancer [78–
80]. In some studies, a TH2-like response (that is, an immune response dominated by IL-4,
IL-10, and IL-13) is associated with tumor progression. While it may be obvious that a TH1
response would be desirable (after all, TH1 responses are associated with effective cytotoxic
T lymphocyte responses), the action of TH2 mediators on the tumor may be more complex
than simply the functional effects on T cell responses. To determine whether the immunosup-
pression observed in TILs from individuals with colorectal cancer and melanoma is permanent
or if it can be reversed given the correct microenvironment, TILs from human melanomas
and colorectal carcinomas were isolated and cocultured in vitro with IL-2, a T cell growth
factor [78]. After 41-48 days of culture Fas levels remained unchanged, but there was a
significant increase in the levels of CD3ε and CD3ζ after extended culture with IL-2. Perforin
levels also increased, suggesting that these cells had an enhanced cytotoxic activity. This
was supported by in vitro assays, which demonstrated an increase in killing by these
cells [78]. These data demonstrate that unresponsive TILs have the potential to have their
immune functions restored given the proper microenvironment, and provide hope that, with
further study, this same restoration of function can one day be achieved in vivo.
Using a human colorectal carcinoma cell line, Kanai et al. [81] examined the effects of
exposure to either recombinant IL-4 or IL-13 on cancer cell-cell adhesion. These mediators
downregulated expression of both E-cadherin and carcinoembryonic antigen, molecules in-
volved in cell-cell adhesion. Altered E-cadherin expression has been correlated with the
differentiation state of the tumor [79,80]. Both IL-4 and IL-13 were produced by freshly
isolated TILs from colorectal cancer patients. Interleukin-4 and IL-13 were also produced
by intraepithelial lymphocytes from control tissues [81]. If one considers the role of soluble
mediators in tumor cell adhesiveness, then it is apparent that the tumor cell has adapted
to the local immune processes of the host, and exploited the available cytokines for its
own purposes.
Cytotoxic T cells, like CD4⫹ T cells, have been further classified according to the
cytokines that they produce upon activation. T cytotoxic-1 cells produce IL-2 and interferon-γ
(IFN-γ); T cytotoxic-2 cells produce IL-4, IL-5, and IL-6. In a study of TILs isolated from
human cervical cancer tissue, activated T cells were determined to be primarily of the TH2/Tc2
cytotoxic-2 phenotype, producing high levels of IL-4 and IL-5, and low levels of interferon-γ
[82]. TGF-β and IL-10 were also produced by cervical cancer cells, but not normal cervical
epithelial cells because the tumor itself has the capacity to produce mediators that directly
impact TIL development. In vitro studies demonstrated that production of cytokines typi-
cally considered to be immunosuppressive (ie, IL-10 and TGF-β) drive development of the
TILs towards a TH2/Tc2 cytotoxic-2 phenotype [82]. This finding may play a significant
role in understanding how tumors escape from immunosurveillance; that is, they produce
modulatory proteins that dampen the host immune response.
Von Bernstorff et al. [83] assayed serum levels of 3 immunosuppressive cytokines IL-10,
TGF-β1, and TGF-β2 in patients with pancreatic cancer, hypothesizing that these mediators
would be increased in individuals with tumors compared to controls. Indeed, all 3 soluble
 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166 161

mediators were significantly increased in pancreatic cancer patients compared with healthy
controls. A caveat to these studies was that no significant differences were found between cancer
patients and individuals with pancreatitis and the authors did not note a correlation between
disseminated disease and elevated cytokine levels [83]. The finding that there was no differ-
ence between pancreatic cancer patients and patients with pancreatitis poses an interesting
point to the use of serum soluble mediator levels as markers of disease—is it really the
cytokine that is important, or is it more crucial to know how long the proteins have been
upregulated for? Presumably patients with pancreatitis are experiencing a different type of
immunosuppression (related to treatment with corticosteroids and/or alterations in cytokine
levels) compared with those with pancreatic cancer (related to loss of T cell responses).

7. Gamma delta cells

To this point, we have focused on the role of α/β T cells in immune responses to cancer,
because the functions of this subset of T cells are better defined than those of γ/δ T cells;
α/β T cells also comprise most T lymphocytes in the body (⬎90%). A small number of
studies focused on the contribution of γ/δ T cells, a relatively minor T cell population, in
the immune response to cancer. γ/δ Cells recognize native (that is, unprocessed) antigen in the
context of nonclassical MHC molecules such as MICA and MICB, which are normally
limited to the intestinal epithelium. Work by Groh et al. [19] has localized MICA and MICB
expression to cancers of the ovary, lung, kidney, breast, prostate, and colon. In addition to
increased expression of nonclassical MHC molecules, these carcinomas also had increased
levels of γ/δ T cells in their tumor infiltrates. The significance of these findings in clinical
utility remains undefined at this time, but merits further study.

8. Summary
Within this review, we have attempted to address some of the parameters that have been
studied over the last few years and correlated with patient prognosis in various forms of
cancer. Given the sheer volume of studies, we could obviously not even begin to address
all of the available data. Our purpose was rather, to present some of the general hypotheses
that are being examined in the field of TILs at this time, with a focus on the adaptive
immune response. Because of the contradictory data one may consider the review and be
left wondering, do we really know more than we did 30 years ago? Does what we now
know really advance the field of cancer etiology and the role of immunotherapy? We would
have to answer these questions yes. While many of the highly experimental clinical trials have
not been an overwhelming success, there has been progress. What have we learned? Perhaps
one of the most valuable lessons from the past 30 years of TIL study is that some of the
contrary results are providing insight into the role and function of TILs. Differences in how
patients are grouped clearly impacts the results of a study; subdividing patients on particular
genetic characteristics of either the tumor or the patient also allows one to reduce some of
the variation observed in experiments based on clinical samples. For example, data would
be more clear-cut if patients with Lynch Syndrome were considered separately from all
162 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166

nonhereditary colorectal cancer patients. Specifically, Lynch Syndrome patients represent

individuals with a unique prognosis from colorectal cancer patients in general, so why should
we group them together for pathologic analysis?
Another consideration in these kinds of studies is that negative data are probably significant,
at least in some instances. Openness to these findings, both on the part of the investigator and
the reviewer, is needed. It is refreshing to read those works wherein the authors state that their
hypothesis did not hold up and propose an alternative hypothesis at the end of their studies,
and thereby allowing the reader to gain a greater view as to what the true intention of the
study was, and the road that the authors traveled.
The continuum of antitumor responses encompasses a wide range of possibilities for the
host (Fig. 2). The host may die because of an immune response that does not develop
properly; the patient does not generate a strong cytotoxic T cell response, the cells are not
able to enter the tumor site because of a lack of adhesion molecule expression, or a low
level of cytokines that are involved cellular immunity. Alternatively, the tumor may produce
factors that are able to subvert the immune response. The tumor may undermine the
immune response by inducing apoptosis of the responding T cell, immunosuppresses the host,
or using the T cell as a means to metastasize. An understanding of the balance that must be
achieved between all of these immune factors is key to gaining insight into this disease.

Fig. 2. Spectrum of the immune response in the host and the subsequent impact on tumor growth and metastases.
At one end of the spectrum, the host’s immune system responds vigorously to the tumor and is able to mount
an effective immune response, and clear the tumor from the body. Patient prognosis is promising. At the opposite
end of the spectrum, the host is unable to contain tumor growth and control metastases. In this case, patient
prognosis is poor. Between these 2 extremes, the host mounts an intermediate immune response and is able to
contain the tumor to its site of origin.
 K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166 163

Acknowledgments
The authors thank the American Cancer Society (RSG-01-250-01-MBC; KMD), the Na-
tional Institutes of Health (5 U01 CA086389-04; HTL), and the Nebraska Cancer and
Smoking Disease Research Program (LB595; KMD, HTL) for their support.

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