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

Tumor infiltrating lymphocytes (TILs): Lessons learnedin 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 factorassociated 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 cancerappear 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 andmetastases. Animal models of cancer, imperfect as they are, can provide investigators with modelsystems 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 inhumans because of differences in surface molecule expression and signaling pathways between thespecies, 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: [email protected] (K.M. Drescher)

1529-1049/05/$ – see front matter 2005 Elsevier Inc. All rights reserved.

doi: 10.1016/j.cair.2005.03.002

mailto:[email protected]







K.M. Drescher, H.T. Lynch/Clin. Applied Immunol. Rev. 5 (2005) 149–166 150

immunotherapy. This review will discuss some of the immune parameters studied in tumor

infiltrating lymphocytes and how they have been associated with patient prognosis.

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 rankssecond only to heart disease as the cause of death in Americans [1]. While death rates dueto heart disease have dropped precipitously since 1950, deathsrates dueto cancerhave remained

relatively steady. The standard approach to treating most forms of cancer consists of surgeryfollowed by radiation and/or chemotherapy; nonetheless, cancer oftenmetastasizes widely, thatis, patients with colorectal cancer frequently develop liver cancer. While approximately 25%of colorectal cancer patients have liver metastases at diagnosis, an additional quarter of theremaining patients develop liver metastases within 2 years of diagnosis [2]. Why some patientsare refractory to developing metastases is unknown, but one hypothesis is that the immunesystem 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 responseagainst tumors, one would assume that the same factors critical to an immune response to anyantigen would be similar to those involved in the body’s ability to control tumors; theseinclude antigenicity of the tumor, ability to recruit an optimal mixture of cell types tothe tumor site, suitable cytokine production by both the tumor and the infiltrating cells, andexpression of cell surface molecules that enhance immune responses. In addition to thesefactors we are now beginning to realize that the tumor, like many bacteria and viruses, hasthe ability to alter the microenvironment to promote survival and dispersement of the tumorcells 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 tumorgrowth and spread via the adaptive immune response originated over 50 years ago with aseries 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 asyngeneic mouse; the mouse that the tumor was originally obtained from resisted tumorgrowth, while the naive syngeneic mouse was unable to prevent tumor growth [3]. Thesestudies clearly indicated that the adaptive immune system can be stimulated to respond toa particular tumor, and that this response is sufficient to reject subsequent challenges with anidentical tumor. Despite the promise of these early data, in vivo the human immune systemdoes 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 bequantified. 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-inducedsarcomas) may have been fortuitous because many tumors are weakly antigenic and expressmodified host proteins on their surface. Conventional wisdom would suggest that whentumors express unique antigens on their surface, the immune system could be stimulated tocontrol their growth and metastases. Our knowledge to date suggests that if such responses




occur in vivo, other mechanisms can be invoked to actively suppress the desired antitumorresponses in many instances. Gene polymorphisms, particularly in genes associated with the

immune response (especially cytok ine genes), have also been associated with host prognosisin different forms of cancer [4–8].

The inherent nature of many tumor antigens makes it difficult for the host to mount astrong antitumor response. Tumor antigens are often aberrantly expressed host proteins thatare the result of developmental dysregulations or small alterations in the normal protein. Asin an immune response to any antigen, these proteins must be present at levels sufficient tostimulate an immune response when presented to T lymphocytes by major histocompatibilitycomplex (MHC) molecules on the surface of an antigen presenting cell (APC). Becausetumor 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 deletedduring thymic selection, or peripheral tolerance is induced if the T cell’s first encounter withpeptide is on the surface of an APC that does not express the correct costimulatory moleculeson its surface. A naive T cell requires interaction with professional APCs (such as dendriticcells or macrophages) for these initial encounters to prevent anergy of the T cell fromoccurring. Proteins unique to immune privileged sites (ie, retina, brain, and testis) are normallysequestered 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 typesof antigens include MAGE-1 and MAGE-3 that are expressed in some forms of breast andbrain cancers [9,10]. These proteins are normally expressed in the testis. Because thetestis is inaccessible to the immune system under normal conditions, T cells reactive topeptides 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 thatindividuals with high levels of inflammatory cells in their tumors had a better prognosisthan those without tumor infiltrates. Since this report, numerous immunohistochemicalstudies 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 thetumor 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 hostwould be provided; by exploiting these naturally occurring control mechanisms, effectiveimmunotherapies could be developed. Unfortunately, the reality of the data from these studieswas less clear-cut than that anticipated in the early work. While patients with increasednumbers 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 thesoluble 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 hostmay have multiple strategies to control or alternatively, fail to control, malignancies. It istherefore not surprising that there is no consensus regarding the type of cells or theirproducts that are required for optimal tumor control. In the case of cervical cancer, in whicha large percentage of the cancers are attributed to infection with human papilloma virus [25],




an effective immune response may be inherently different than cancers caused by a nonviraletiologic 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, itis difficult to predict what other cell types and immune products are important for an effectivedefense in cancers attributed to multiple genetic events, which may or may not be furtherinfluenced by various pathogenic environmental agents.

There is no agreement regarding the utility of examining human tumor specimens forinfiltration by lymphocytes as either a prognostic indicator or a means to guide treatmentoptions for the patient. Individuals with carcinomas bearing particular characteristics, suchas colorectal cancers with high levels of DNA microsatellite instability, have increased levelsof 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 instabilityand TILs, were highly correlated with each other as well as a particular prognosis, physicianscould make targeted treatment decisions based on an examination of a simple pathologysample rather than on requiring more technically demanding and expensive molecularbiology 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 acytotoxic mechanism; TILs may also produce cytokines such as interferon-γ that potentiatesa 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 cellsusually need CD4T cells to function optimally. The ratio of CD4 to CD8T cells is likelya 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 metastaseshad lower levels of CD4 T cells within the tumor than those without metastases, supportinga role for CD4 T cells in tumor control [12], and illustrating the critical balance of cellphenotypes 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 solublemediator (cytokine) production; these cytokines provide CD8 T cells with growth factorsrequired 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 thecharacteristics 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 onthe 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 withantigen, thus making thehost unresponsive to that particular antigen for life. Dendriticcells alsohave the capacity for cross-priming, a process involving presentation of exogenous antigens




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 tumorantigens, because tumors often express low levels of MHC Class I on their surface. Severalstudies have reported that patients with large numbers of infiltrating dendritic cells withintheir 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 tumorsite also had an increased incidence of metastases [31].

Reichert et al. [28] performed immunostaining on samples from patients with primaryoral 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. Aninteresting observation was that TILs within tumors infiltrated with low levels of dendriticcells 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 thesignal to the T cell once ligation of the T cell receptor (TCR) occurs, these TILs cannot




respond appropriately to antigen. Because of this association between the loss of the ζ chainand 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 andT cells, and not solely a function of TIL density. A similar correlation between ζ chainexpression and lymph node metastases has also been noted in a study of patients with gastriccancer [33].

The study of human dendritic cells in colon cancers found that the density of dendriticcells 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 inthe normal colon [34]. This finding suggests that factors within the tumor microenvironmentlimit 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 thelevel 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 conventionalthought—instead of the tumor products actively suppressing infiltration of immune cells, itmay be more fruitful in some instances to examine alterations in proinflammatory mediators.A similar attempt to correlate dendritic cell numbers with survival in patients diagnosedwith pancreatic adenocarcinomas was unsuccessful because of the scarcity of both dendriticcells and lymphocytes [35].

Because of their ability to take up, process, and present antigen, significant efforts are beingfocused on developing immunotherapy approaches that enhance the function of dendritic cells[36,37], with the hypothesis that enhanced dendritic cell function will result in increasedcytotoxic T lymphocyte generation, thereby allowing the host to control tumor growth. Theinfluence of dendritic cells on tumor control and metastases is complicated by the existence of various dendritic cell subsets derived from blood monocytes, hemopoietic precursors, orskin [38]. Many studies have focused on the use of dendritic cells pulsed with antigen as ameans of cancer immunotherapy [39–41]. While these types of studies have been successfulin small animal models, the success and practicality of this approach in clinical medicine

remains to be seen.

3. CD4CD25 T regulatory cells: a balance between tumor responses

and autoimmunity?

Regulatory T cells (T regs) are a population of CD4CD25 T cells that participate inimmune regulation via the suppression of immune responses. This subpopulation of CD4T 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 highlevels of IL-10. In several animal models of autoimmunity, the transcription factor Foxp3has also been designated as a marker of T regs [42]. Regulatory T cells are global suppressorsof immune function, impacting both CD8 T cells and CD4CD25 T cells. In murinemodels of autoimmune disease, in vivo depletion of CD4CD25 T cells increases the level




of organ-specific autoimmunity [43]. While it is accepted that T regs are critical in maintainingself-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 certaininfectious agents or some cancers due to the presence of this CD4CD25 population of T cells?

While TILs are described in many forms of cancer, high levels of TILs are rarely associatedwith a complete cure. One could argue that a partial explanation of this observation isthat cancer patients are frequently immunosuppressed due to the treatment regimens used.However, cancer patients with normal levels of peripheral lymphocytes often have poorrecall 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 restoredantitumor responses in animals formerly unresponsive to the tumor [44]. The animals alsoexperienced 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 modelof progressive breast cancer T regs were detected within 14 days of tumor challenge, andthis population persisted throughout the course of the experiment [44], supporting theview that there is an intrinsic mechanism that operates to suppress the immune responses. Itis significant that these cell types appear early in the immune response to tumors andconsequently, suggests that tumors devise immune evasion strategies almost immediatelyupon development.

Recent work in humans has examined whether patients with cancer have altered levelsof T regs in the periphery compared with healthy individuals, or if there is an associationbetween the level of T regs and cancer stage. Ichihara et al. [45] reported a significant increasein the number of T regulatory cells in the periphery of patients with either gastric or esophagealcancer compared to control subjects. The CD4CD25 cells isolated from gastric cancerpatients secreted IL-10 but not interferon-γ and suppressed proliferation of autologousCD4CD25 T cells, supporting the role of these cells as immune response inhibitors. Inanother study, CD4CD25 T cells from cancer patients also expressed cytotoxic T lympho-cyte antigen-4 (CTLA-4) and CD45RO on their surf ace. The regulatory cells in the periphery

of this set of cancer patients also produced TGF-β [46]. Cytotoxic T lymphocyte antigen-4is a surface molecule associated with inhibition of T cell activation and is also a markerassociated with T regs. Another study found increased levels of CD4CD25 TILs intissue samples from gastric cancer patients compared to those of CD4CD25 normalintraepithelial 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 areclassified as T regulatory cells. Other molecules such as CTLA-4, foxp3, and GITR are alsoexpressed in this subset of T cells, at least in mice. The mechanism of the suppression elicitedby 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 thatcell-cell contact is required [47].

In considering the potential for immunotherapy in the treatment of cancer one must considerthe potential hazards involved in immunizing against tumor antigens. Could immunotherapypotentially do more harm than good without a complete understanding of the factors that




control T reg development? If one can overcome these hurdles and sufficiently suppressT 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 controllingautoimmune responses and enhancing immune responses to tumors is a delicate one, anddisrupting and altering this balance may predispose the host to autoimmune disease becauseimmunotherapy aimed at increased antitumor immunity [48,49]. Could those treated for cancerdevelop equally devastating multiorgan autoimmune diseases because of immunotherapy? Anunderstanding of the development, functions, and control of T regs remains critical to ourability 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/granzymepathway or the Fas/Fas ligand (FasL) pathway. The end result of both pathways is apoptosisof 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 lymphocytescan express both Fas and FasL on their surface. While it is obvious that it would be desirablefor cytotoxic T cells to express FasL on their surface, Fas expression by the T cell has 2potential roles: first, ligation of Fas on the T cell induces apoptosis of the cell, which resolvesan immune response once an antigen has been cleared from the host; second, Fas expressionby 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 humantissue suggest that some cancers have exploited this second mechanism to subvert the immuneresponse 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 signalto a Fas-bearing tumor cell and the tumor cell undergoes apoptosis. Despite infiltration of thetumor site with an optimal ratio of CD4 /CD8 T cells with the correct TCR, TILs maynot control tumor growth and/or metastases in this scenario. Several studies using humantissue 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 delivera death signal to the tumor cell, resulting in apoptosis of the Fas-bearing tumor cell. Expressionof membrane-bound FasL on the tumor cell would lead to the opposite effect, d eletion of the TIL. This process of immune evasion has been termed Fas counterattack [15] anddescribed 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 studiescorrelating 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, frequentlyhigh 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 considerthe level of apoptotic TILs as well as FasL expression by the tumor cells to accuratelypredict survival [15,53,55,61].

A recent study by Asanuma et al. [62] has provided insight into a potential mechanismas to how FasL-mediated immunosuppression is induced. The role of survivin, a member




of the inhibitor-of-apoptosis family, in Fas counterattack was examined. Immunostainingexperiments using human colon cancer tissue demonstrated a correlation between staining

intensity for survivin and FasL. Transfection of LS180 cells with a plasmid encodingsurvivin resulted in increased FasL expression on the cells’ surface. Subsequent transfectionof these cells with survivin-specific small inhibitory RNA downregulated FasL expressionsuggesting that survivin is a key contributor to FasL induction by tumor cells. While it ispremature to predict whether survivin-mediated upregulation of FasL will represent a generalmechanism used by a broad array of cancers to control the host immune response, it doespotentially 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 allograftrejection the authors hypothesized that in the absence of TGF-β1 FasL expression triggeredneutrophil recruitment, which would be detrimental to tumor growth and/or survival [64].No correlation between FasL expression in colon cancer, neutrophil recruitment, and TGF-β1expression was found in tissues from human colon cancer patients, although FasL wascorrelated 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 apoptoticTILs. The authors reported that apoptotic TILs most often localized to tumors classified asmicrosatellite stable. Additionally, a correlation between the number of apoptotic TILs andtumor 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 studyreported that tumors with the highest FasL levels were categorized as MSI-High; MSI-Highcancers were those with a more favorable prognosis [52]. Perhaps one of the most importantfindings to come out of this study was that microsatellite stable tumors had the highest levelof apoptotic TILs but not the high levels of membrane-bound FasL, implying that anothermechanism that does not involve membrane-bound FasL is involved in TIL apoptosis. Analternative mechanism could also invoke processes that protect TILs from apoptotic cell

death. Other studies have examined FasL expression in both primary and metastaticcolorectal 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 prognosticindicator 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 inpatients with a poor prognosis. To further understand the relationship between these proteinsand 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 cellsexperienced a 2- to 3-fold increase in Bcl-x protein compared to control cells; increasedmessenger RNA levels were also observed. Immunodeficient mice were then injected withthe c-myb overexpressing cells. Tumors from mice injected with the c-Myb–transfected cellswere 2.5 times larger than tumors from mice injected with control-transfected cells. Further,




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 interactionsmay not overcome resistance of the tumor cells to apoptosis.

5. Adhesion molecules

The significance of cellular adhesion molecule expression as a prognostic indicator incancer is less well defined than many of the other factors discussed thus far. The argumentsdetailing 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 interactswith intracellular adhesion molecule-1 or -2 (ICAM-1, -2) on the APC. This interactionprovides stability between the APC and the responding T cell, allowing appropriate signalsto be delivered from the APC to the T cell via the TCR. It is hypothesized that this interactionbetween 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 absenceof these interactions, effective T cell signaling cannot occur.

Early work on human melanomas correlated increased adhesion molecule expression withan increased risk of metastases in the patients [66]. While the rationale for this finding isnot intuitive, the authors offered an intriguing hypothesis. They proposed that ligation betweenICAM-1 positive tumor cells and LFA-1 positive T cells allowed the tumor cell to disseminateto 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 patientswith metastases compared to patients without metastases; patients with ICAM-1 negativetumors ha d a significantly worse prognosis compared with patients whose tumors were ICAM-positive [67]. Alternatively, other studies on cancer patients have found increased ICAM-1expression in metastatic melanoma lesions [47,68], as well as an association between increasedtumor size and metastases in gastric cancer [69]. One could easily envision a scenario whereincreased 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-1expression, an appropriate signal would not be generated through the TCR. These high levelsof ICAM-1 expression may also come with a risk. While increased adhesion to T cells wouldenhance antigen presentation, this increased adhesion to the T cells may also allow tumor cellsto 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 criticalto controlling a certain type of tumor. If high ICAM-1 expression were found to be ageneral 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 antigens1-6, 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




TILs had decreased LFA-1 on their surface compared to the levels detected on peripheralblood 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 reducedon both CD4 and CD8 TILs, compared to the levels observed in the peripheralblood lymphocytes. Functionally, the CD8 TILs exhibited decreased adherence to bothICAM-1 and vascular cell adhesion molecule-1–coated substrates compared with PBLs. Inaddition, 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 towhy the TILs failed to lyse the tumors. Without the ability to make appropriate contactbetween the APC (whether it be on the dendritic cell presenting exogenous antigen or thetumor 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

fewerrelapsesandbettersurvivalcomparedtopatientswithlowTILlevels[71]. Correlatedwiththis finding was the observation that patients with diffuse large-cell lymphoma with low TILlevels also had reduced expression of MHC Class I and Class II within the tumor. Furtherexamination of these patients for HLA, ICAM, LFA-1, ICAM-1, B7.1, and B7.2 expressionfollowed by categorizing the patients into subgroups based on TIL levels determined thatall patients with low levels of TILs were deficient in expression of at least one of the surfacemolecules under study [72]. These data also indicate that minimally, one portion of the antigenrecognition and presentation process was lacking. While these data are not in agreementwith some other studies on diffuse large-cell lymphoma [73,74], the differences may beattributed 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 effectiveimmune function have been associated with metastatic events. These data challenge the ideaof one-size-fits-all treatment. Adhesion molecule expression may be a marker that wouldbenefit from monitoring throughout the course of treatment and adjustments to therapy canbe made accordingly.

6. Cytokines

The microenvironment, particularly the soluble mediators expressed, is known to impactimmune cell function. Likewise, the local cytokine milieu in the tumor is also correlated withthe immune response to the tumor. Transforming growth factor-β (TGF-β) is a multifunctionalmediator critical to the development of B, T, and natural killer cells; it is also involved inthe 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 cellshad lower levels of TGF-βl [75]. Consistent with these data, in vitro studies have shownthat treatment with TGF-βl decreased Fas expression and susceptibility to apoptosis in bothhuman dendritic cell precursors [76] and murine bone marrow progenitor cells [77]. Together,




these data suggest that one potential mechanism for tumor evasion of the immune responseinvolves 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 TH1response would be desirable (after all, TH1 responses are associated with effective cytotoxicT lymphocyte responses), the action of TH2 mediators on the tumor may be more complexthan 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 permanentor 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 growthfactor [78]. After 41-48 days of culture Fas levels remained unchanged, but there was asignificant increase in the levels of CD3ε and CD3ζ after extended culture with IL-2. Perforinlevels also increased, suggesting that these cells had an enhanced cytotoxic activity. Thiswas supported by in vitro assays, which demonstrated an increase in killing by thesecells [78]. These data demonstrate that unresponsive TILs have the potential to have theirimmune functions restored given the proper microenvironment, and provide hope that, withfurther 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 mediatorsdownregulated expression of both E-cadherin and carcinoembryonic antigen, molecules in-volved in cell-cell adhesion. Altered E-cadherin expression has been correlated with thedifferentiation state of the tumor [79,80]. Both IL-4 and IL-13 were produced by freshlyisolated TILs from colorectal cancer patients. Interleuk in-4 and IL-13 were also producedby intraepithelial lymphocytes from control tissues [81]. If one considers the role of solublemediators in tumor cell adhesiveness, then it is apparent that the tumor cell has adaptedto the local immune processes of the host, and exploited the available cytokines for itsown 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 fromhuman cervical cancer tissue, activated T cells were determined to be primarily of the TH2/Tc2cytotoxic-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 cervicalepithelial cells because the tumor itself has the capacity to produce mediators that directlyimpact 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 theTILs 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 producemodulatory 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 mediatorswould be increased in individuals with tumors compared to controls. Indeed, all 3 soluble




mediators were significantly increased in pancreatic cancer patients compared with healthycontrols. A caveatto these studies wasthat no significant differences were found between cancer

patients and individuals with pancreatitis and the authors did not note a correlation betweendisseminated disease and elevated cytokine levels [83]. The finding that there was no differ-ence between pancreatic cancer patients and patients with pancreatitis poses an interestingpoint to the use of serum soluble mediator levels as markers of disease—is it really thecytokine that is important, or is it more crucial to know how long the proteins have beenupregulated for? Presumably patients with pancreatitis are experiencing a different type of immunosuppression (related to treatment with corticosteroids and/or alterations in cytokinelevels) 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, inthe immune response to cancer. γ / δ Cells recognize native (that is, unprocessed) antigen in thecontext of nonclassical MHC molecules such as MICA and MICB, which are normallylimited 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 toincreased expression of nonclassical MHC molecules, these carcinomas also had increasedlevels of γ / δ T cells in their tumor infiltrates. The significance of these findings in clinicalutility 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 beenstudied 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 addressall of the available data. Our purpose was rather, to present some of the general hypothesesthat are being examined in the field of TILs at this time, with a focus on the adaptiveimmune response. Because of the contradictory data one may consider the review and beleft wondering, do we really know more than we did 30 years ago? Does what we nowknow really advance the field of cancer etiology and the role of immunotherapy? We wouldhave to answer these questions yes. While many of the highly experimental clinical trials havenot been an overwhelming success, there has been progress. What have we learned? Perhapsone 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 howpatients are grouped clearly impacts the results of a study; subdividing patients on particulargenetic 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 wouldbe more clear-cut if patients with Lynch Syndrome were considered separately from all




nonhereditary colorectal cancer patients. Specifically, Lynch Syndrome patients representindividuals 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 andthe reviewer, is needed. It is refreshing to read those works wherein the authors state that theirhypothesis 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 thestudy was, and the road that the authors traveled.

The continuum of antitumor responses encompasses a wide range of possibilities for thehost (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 notable to enter the tumor site because of a lack of adhesion molecule expression, or a lowlevel of cytokines that are involved cellular immunity. Alternatively, the tumor may producefactors that are able to subvert the immune response. The tumor may undermine theimmune 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 beachieved 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.




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 andSmoking Disease Research Program (LB595; KMD, HTL) for their support.

References

[1] American Cancer Society Website. 2004.

[2] Hughes KS. Resection of the liver for colorectal-carcinoma metastases: a multi-institutional study of

indications for resection. Surgery 1988;103:278–88.

[3] Foley EJ. Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer

Res 1953;13:835–7.[4] Smith KC, Bateman AC, Fussell HM, Howell WM. Cytokine gene polymorphisms and breast cancer

susceptibility and prognosis. Eur J Immunogenet 2004;31:167–73.

[5] Loktionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer Lett 2004;208:1–33.

[6] Martin AM, Athanasiadis G, Greshock JD, Fisher J, Lux MP, Calzone K, et al. Population frequencies of

single nucleotide polymorphisms (SNPs) in immuno-modulatory genes. Hum Hered 2003;55:171–8.

[7] Nakamura E, Megumi Y, Kobayashi T, Kamoto T, Ishitoya S, Terachi T, et al. Genetic polymorphisms of

the interleukin-4 receptor alpha gene are associated with an increasing risk and a poor prognosis of sporadic

renal cell carcinoma in a Japanese population. Clin Cancer Res 2002;8:2620–5.

[8] Calhoun ES, McGovern RM, Janney CA, Cerhan JR, Iturria SJ, Smith DI, et al. Host genetic polymorphism

analysis in cervical cancer. Clin Chem 2002;48:1218–24.

[9] Brasseur F, Marchand M, Vanwijck R, Herin M, Lethe B, Chomez P, et al. Human gene MAGE-1, whichcodes for a tumor-rejection antigen, is expressed by some breast tumors. Int J Cancer 1992;52:839–41.

[10] Sahin U, Koslowski M, Tureci O, Eberle T, Zwick C, Romeike B, et al. Expression of cancer testis genes

in human brain tumors. Clin Cancer Res 2000;6:3916–22.

[11] Murray D, Hreno A, Dutton J, Hampson LG. Prognosis in colon cancer: a pathologic reassessment. Arch

Surg 1975;110:908–13.

[12] Sheu BC, Hsu SM, Ho HN, Lin RH, Torng PL, Huang SC. Reversed CD4/CD8 ratios of tumor-infiltrating

lymphocytes are correlated with the progression of human cervical carcinoma. Cancer 1999;86:1537–43.

[13] Mann B, Gratchev A, Bohm C, Hanski ML, Foss HD, Demel G, et al. FasL is more frequently expressed

in liver metastases of colorectal cancer than in matched primary carcinomas. Br J Cancer 2002;79:1262–9.

[14] Okada K, Komuta K, Hashimoto S, Matsuzaki S, Kanematsu T, Koji T. Frequency of apoptosis of tumor-

infiltrating lymphocytes induced by fas counterattack in human colorectal carcinoma and its correlation

with prognosis. Clin Cancer Res 2000;6:3560–4.

[15] Bennett MW, O’Connell J, O’Sullivan GC, Brady C, Roche D, Collins JK, et al. The fas counterattack

in vivo: apoptotic depletion of tumor-infiltrating lymphocytes associated with fas ligand expression by

human esophageal carcinoma. J Immunol 1998;160:5669–75.

[16] Fisk B, Anderson BW, Gravitt KR, O’Brian CA, Kudelka AP, Murray JL. Identification of naturally

processed human ovarian peptides recognized by tumor-associated CD 8 cytotoxic T lymphocytes. Cancer

Res 1997;57:87–93.

[17] Linehan DC, Goedegebuure PS, Peoples GE, Rogers SO, Eberlein TJ. Tumor-specific and HLA-A2-restricted

cytolysis by tumor associated lymphocytes in human metastatic breast cancer. J Immunol 1995;155:4486–91.

[18] Puccetti L, Manetti R, Parronchi P, Piccinni MP, Mavilia C, Carini M, et al. Role of low nuclear grading

of renal carcinoma cells in the functional profile of tumor-infiltrating T cells. Int J Cancer 2002;98:674–81.

[19] Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein K, Spies T. Broad tumor-associated expression and

recognition by tumor-derived γδ T cells of MICA and MICE. Proc Natl Acad Sci U S A 1999;96:6879–84.

[20] Michael-Robinson JM, Biemer-Huttmann AE, Purdie DM, Walsh MD, Simms LA, Biden KG, et al. Tumour

infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to

microsatellite instability status. Gut 2001;48:360–6.




[21] Calhoun RF II, Naziruddin B, Enriquez-Rincon F, Duffy BF, Ritter JM, Sundaresan S, et al. Evidence for

cytotoxic T lymphocyte response against human lung cancer: Reconstitution of antigenic epitope with

peptide eluted from lung adenocarcinoma MHC class I. Surgery 2000;128:76–85.[22] Schumacher K, Haensch W, Roefzaad C, Schlag PM. Prognostic significance of activated CD8 T cell

infiltrations within esophageal carcinomas. Cancer Res 2001;61:3932–6.

[23] Fukuzawa Y, Takahashi K, Furuta K, Tagaya T, Ishikawa T, Wada K, et al. Expression of Fas/Fas ligand (FasL)

and its involvement in infiltrating lymphocytes in hepatocellular carcinoma. J Gastroenterol 2001;36:681–8.

[24] Zietz C, Rumpler U, Sturzl M, Lohrs U. Inverse relation of fas-ligand and tumor-infiltrating lymphocytes

in angiosarcanoma. Am J Pathol 2001;159:963–70.

[25] Murthy NS, Mathew A. Risk factors for pre-cancerous lesions of the cervix. Eur J Cancer Prev 2000;9:5–14.

[26] Lothe RA, Peltomaki P, Meling GI. Genomic instability in colorectal cancer: relationship to clinicopathologi-

cal variables and family history. Cancer Res 1993;53:5849–52.

[27] Wright CM, Cent OF, Barker M. The prognostic significance of extensive microsatellite instability in

sporadic clinicopathological stage C colorectal cancer. Br J Surg 2000;87:1197–202.[28] Reichert TE, Scheuer C, Day R, Wagner W, Whiteside TL. The number of intratumoral dendritic cells

and ζ-chain expression in T Cells as prognostic and survival biomarkers in patients with oral carcinoma.

Cancer 2001;91:2136–47.

[29] Zeid NA, Muller HK. S100 positive dendritic cells in human lung tumors associated with cell differentiation

and enhanced survival. Pathology 1993;25:338–43.

[30] KenjiA, MasakiM, Munetomo E. S-100protein-positive dendritic cellsin colorectaladenocarcinomas.Cancer

1989;63:496–503.

[31] Tsujitani S, Kakeji Y, Maehara Y, Sugimachi K, Kaibara N. Dendritic cells prevent lymph node metastasis

in patients in gastric cancer. In Vivo 1993;7:233–7.

[32] Goldman SA, Baker E, Weyant RJ, Clarke MR, Myers JN, Lotze MT. Peritumoral CD1a-positive dendritic

cells are associated with improved survival in patients with tongue carcinoma. Arch Otolaryngol HeadNeck Surg 1998;124:641–6.

[33] Ishigama S, Natsugoe S, Tokuda K, Nakajo A, Higasi H, Iwashige H, et al. CD3-ζ chain expression of

intratumoral lymphocytes is closely related to survival in gastric carcinoma patients. Cancer 2002;94:

1437–42.

[34] Schwaab T, Weiss JE, Schned AR, Barth RJ. Dendritic cell infiltration in colon cancer. J Immunother

2001;24:130–7.

[35] Dallal RM, Christakos P, Lee K, Egawa S, Son YI, Lotze MT. Paucity of dendritic cells in pancreatic

cancer. Surgery 2002;131:135–8.

[36] Tanaka F, Hashimoto W, Okamura H, Robbings PD, Lotze MT, Tahara H. Rapid generation of potent and

tumor-specific cytotoxic T lymphocytes by interleukin 18 using dendritic cells and natural killer cells.

Cancer Res 2000;60:4838–44.

[37] Tatsumi T, Huang J, Gooding WE, Gambotto A, Robbins PD, Vujanovic NL, et al. Intratumoral delivery

of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant

disease in association with broadly reactive Tc1-type immunity. Cancer Res 2003;2003:6378–86.

[38] Suzuki A, Masuda A, Nagata H, Kameoka S, Kikawada Y, Yamakawa M, et al. Mature dendritic cells

make clusters with T cells in the invasive margin of colorectal carcinoma. J Pathol 2002;196:37–43.

[39] Wang HY, Fu T, Wang G, Zeng G, Perry-Lalley DM, Yang JC, et al. Induction of CD4 T cell-dependent

antitumor immunity by TAT-mediated tumor antigen delivery into dendritic cells. J Clin Invest 2002;

109:1463–70.

[40] Sloan JM, Kershaw MH, Touloukian CE, Lapointe R, Robbins PF, Restifo NP, et al. MHC class I and class

II presentation of tumor antigen in retrovirally and adenovirally transduced dendritic cells. Cancer Gene

Ther 2002;9:946–50.

[41] Kershaw MH, Hsu C, Mondesire W, Parker LL, Wang G, Overwijk WW, et al. Immunization against

endogenous retroviral tumor-associated antigens. Cancer Res 2001;61:7920–4.

[42] Chen W, Jin W, Hardegen N, Lei K-J, Li L, Marinos N, et al. Conversion of peripheral CD4CD25- naive

T cells to CD4CD25 regulatory T cells by TGF-β induction of transcription factor of Foxp3. J Exp

Med 2003;198:1875–86.




[43] Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated

T cells expressing IL-2 receptor α-chains (CD25). Breakdown of a single mechanism of self-tolerance

causes various autoimmune diseases. J Immunol 1995;155:1151–64.[44] Shimizu J, Yamazaki S, Sakaguchi S. Induction of tumor immunity by removing CD25CD4 T cells:

a common basis between tumor immunity and autoimmunity. J Immunol 1999;163:5211–8.

[45] Ichihara F, Kono K, Takahashi A, Kawaida H, Sugai H, Fujii H. Increased populations of regulatory

T cells in peripheral blood and tumor-infiltrating lymphocytes in patients with gastric and esophageal

cancers. Clin Cancer Res 2003;9:4404–8.

[46] Wolf AM, Wolf D, Steurer M, Gastl G, Gunsilius E, Grubeck-Loebenstein B. Increase of regulatory T cells

in the peripheral blood of cancer patients. Clin Cancer Res 2003;9:606–12.

[47] Thornton AM, Shevach EM. CD25 immunoregulatory T cells suppress polyclonal T cell activation

in vitro and by inhibiting interleukin 2 production. J Exp Med 1998;188:289–96.

[48] Nelson BH. IL-2, regulatory T cells, and tolerance. J Immunol 2004;172:3983–8.

[49] Wei WZ, Morris GP, Kong YC. Anti-tumor immunity and autoimmunity: a balancing act of regulatoryT cells. Cancer Immunol Immunother 2004;53:73–8.

[50] Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA. Fas ligand-induced apoptosis as a mechanism

of immune privilege. Science 1995;270:1189–92.

[51] Restifo NP. Not so Fas: re-evaluating the mechanisms of immune privilege and tumor escape. Nat

Med 2000;6:493–4.

[52] Michael-Robinson JM, Pandeya N, Cummings MC, Walsh MD, Young JP, Leggett BA, et al. Fas ligand

and tumour counter-attack in colorectal cancer stratified according to microsatellite instability status.

J Pathol 2003;201:46–54.

[53] Hahne M, Rimoldi D, Schroter M, Romero P, Schreier M, French LE, et al. Melanoma cell expression of

Fas (APO-1/CD95) ligand: implications for tumor escape. Science 1996;274:1363–6.

[54] Lim SC. Fas-related apoptosis in gastric adenocarcinoma. Oncol Rep 2003;10:57–63.[55] Bodey B, Bodey B Jr, Siegel SE, Kaiser HE. Fas (APO-1, CD95) receptor expression and new options for

immunotherapy in childhood medulloblastomas. Anticancer Res 1999;19:3293–314.

[56] O’Connell J, O’Sullivan GC, Collins JK, Shanahan F. The Fas counterattack: Fas-mediated T cell killing

by colon cancer cells expressing Fas ligand. J Exp Med 1996;184:1075–82.

[57] O’Connell J, Bennett MW, O’Sullivan GC, Roche D, Kelly J, Collins JK, et al. Fas ligand expression in

primary colon adenocarcinomas: evidence that the Fas counterattack is a prevalent mechanism of immune

evasion in human colon cancer. J Pathol 1998;186:240–6.

[58] Liu C, Chiu JH, Chin T, Wang LS, Li AF, Chow KC, et al. Expression of fas ligand on bile ductule

epithelium in biliary atresia—a poor prognostic factor. J Pediatr Surg 2000;35:1591–6.

[59] Yoong KF, Afford SC, Randhawa S, Hubscher SG, Adams DH. Fas/Fas ligand interaction in human

colorectal hepatic metastases. Am J Pathol 1999;154:693–703.[60] O’Connell J, Bennett MW, O’Sullivan GC, O’Callaghan J, Collins JK, Shanahan F. Expression of fas

(CD95/APO-1) ligand by human breast cancers: significance for tumor immune privilege. Clin Diagn Lab

Immunol 1999;6:457–63.

[61] Bennett MW, O’Connell J, O’Sullivan GC, Roche D, Brady C, Kelly J, et al. Expression of fas ligand

by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer. Gut

1999;44:156–62.

[62] Asanuma K, Tsuji N, Endoh T, Yagihashi A, Watanabe N. Survivin enhances fas ligand expression via

up-regulation of specificity protein 1-mediated gene transcription in colon cancer cells. J Immunol

2004;172:3922–9.

[63] Houston A, Bennett MW, O’Sullivan GC, Shanahan F, O’Connell J. Fas ligand mediates immune privilege

and notinflammation in human colon cancer, irrespective of TGF-β expression.Br J Cancer 2003;89:1345–51.

[64] Chen JJ, Sun Y, Nabel GJ. Regulation of the proinflammatory effects of Fas ligand (CD95). Science

1998;282:1714–7.

[65] Biroccio A, Benassi B, D’Agnano I, D’Angelo C, Buglioni S, Mottolese M, et al. c-Myb and Bcl-x

overexpression predicts poor prognosis in colorectal cancer. Am J Pathol 2001;158:1289–99.




[66] Johnson JP, Stade BG, Holzmann B, Schwable W, Riethmuller G. De novo expression of intercellular-

adhesion molecule 1 in melanoma correlates with increased risk of metastasis. Proc Natl Acad Sci U S A

1989;86:641–4.[67] Maeda K, Kang SM, Sawada T, Nishiguchi Y, Yashiro M, Ogawa Y, et al. Expression of intercellular

adhesion molecule-1 and prognosis in colorectal cancer. Oncol Rep 2002;9:511–4.

[68] Natali PG, Hamby CV, Felding-Habermann B, Liang B, Nicotra MR, DiFilippo F, et al. Clinical significance

of alpha(v)beta3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma

lesions. Cancer Res 1997;57:1554–60.

[69] Koyama S, Ebihara T, Fukao K. Expression of intercellular adhesion molecule 1 (ICAM-1) during the

development of invasion and/or metastasis of gastric carcinoma. J Cancer Res Clin Oncol 1992;118:609–14.

[70] Kitayama J, Nagawa H, Nakayama H, Tuno N, Shibata Y, Muto T. Functional expression of β1 and β2

integrins on tumor infiltrating lymphocytes (TILs) in colorectal cancer. J Gastroenterol 1999;34:327–33.

[71] Lippman SM, Spier CM, Miller TP, Slymen DJ, Rybski JA, Grogan TM. Tumor-infiltrating T-lymphocytes

in B-cell diffuse large cell lymphoma related to disease course. Mod Pathol 1990;3:361–7.[72] Stopeck AT, Gessner A, Miller TP, Hersh EM, Johnson CS, Cui H, et al. Loss of B7.2 (CD86) and

intracellular adhesion molecule 1 (CD54) expression is associated with decreased tumor-infiltrating T

lymphocytes in diffuse B-cell large-cell lymphoma. Clin Cancer Res 2000;6:3904–9.

[73] Horst E, Meijer CJLM, Radaszkiewic T, Ossekopple GJ, van Krieken JHJM, Pals ST. Adhesion molecules

in the prognosis of diffuse large-cell lymphoma: expression of a lymphocyte homing receptor (CD44),

LFA-1 (CD11a/18), and ICAM-1 (CD54). Leukemia 1990;4:595–9.

[74] Medeiros LJ, Weiss LM, Picker LJ, Clayberger C, Horning SJ, Krensky AM, et al. Expression of LFA-1

in non-Hodgkin’s lymphoma. Cancer 1989;63:255–9.

[75] Yuen MF, Hughes RD, Heneghan MA, Langley PG, Norris S. Expression of fas antigen (CD95) in peripheral

blood lymphocytes and in liver-infiltrating, cytotoxic lymphocytes in patients with hepatocellular carcinoma.

Cancer 2001;92:2136–41.[76] Riedl E, Strobl H, Majdic O, Knapp W. TGF-β1 promotes in vitro generation of dendritic cells by protecting

progenitor cells from apoptosis. J Immunol 1997;158:1591–7.

[77] Dybedal I, Guan F, Borge OJ, Veiby OP, Ramsfjell V, Nagata S, et al. Transforming growth factor-β1

abrogates fas-induced growth suppression and apoptosis of murine bone marrow progenitor cells. Blood

1997;90:2284–3403.

[78] De Paola F, Riccobon A, Flamini E, Barzanti F, Granato AM, Mordenti GL, et al. Restored T-cell activation

mechanisms in human tumor-infiltrating lymphocytes from melanomas and colorectal carcinomas after

exposure to interleukin-2. Br J Cancer 2003;88:320–6.

[79] Tamm I, Kikutami T, Cardinale I, Krueger JG. Cell-adhesion-disrupting action of interleukin-6 in human

ductal breast carcinoma cells. Proc Natl Acad Sci U S A 1994;91:3329–33.

[80] Tamm I, Cardinale I, Kikutami T, Krueger JG. E-cadherin distribution in interleukin 6-induced cell-cell

separation of ductal breast carcinoma cells. Proc Natl Acad Sci U S A 1994;91:4338–42.

[81] Kanai T, Watanabe M, Hayashi A, Nakazawa A, Yajima T, Okazawa A, et al. Regulatory effect of interleukin-4

and interleukin-13 on colon cancer cell adhesion. Br J Cancer 2000;82:1717–23.

[82] Sheu BC, Lin RH, Lien HC, Ho HN, Hsu SM, Huang SC. Predominant Th2/Tc2 polarity of tumor-infiltrating

lymphocytes in human cervical cancer. J Immunol 2001;167:2972–8.

[83] von Bernstorff W, Voss M, Freichel S, Schmid A, Vogel I, Johnk C, et al. Systemic and local immunosup-

pression in pancreatic cancer patients. Clin Cancer Res 2001;7:925s–32s.

tumor in flit rating lymphocytes

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