HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Radoja, S. Articles by Frey, A. B. Search for Related Content PubMed PubMed Citation Articles by Radoja, S. Articles by Frey, A. B.

CD8⁺ Tumor-Infiltrating Lymphocytes Are Primed for Fas-Mediated Activation-Induced Cell Death But Are Not Apoptotic In Situ¹

Sasa Radoja^*,, Masanao Saio^*, and Alan B. Frey^2,*

^* Department of Cell Biology and Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016; Institute of Molecular Genetics and Genetic Engineering, Belgrade, Yugoslavia; and Second Department of Pathology, Gifu University School of Medicine, Gifu, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of Fas-mediated activation-induced cell death in antitumor T cells has been hypothesized to permit tumor escape from immune destruction. Several laboratories have proposed that expression of Fas ligand (L) by tumor is the basis for this form of T cell tolerance. In this study, we characterized murine tumor-infiltrating lymphocytes (TIL) for activation status, cell cycle status, level of apoptosis, cytokine secretion, and proliferative capacity. TILs express multiple activation markers (circa CD69, CD95L, CD122, and LFA-1) and contain IL-2 and IFN- mRNAs, but are neither cycling nor apoptotic in situ. In addition, TIL are dramatically suppressed in proliferative response and do not secrete IL-2 and IFN-. However, upon purification and activation in vitro, TIL secrete high levels of IL-2 and IFN-, enter S phase, and then die by Fas-mediated apoptosis. Activation by injection of anti-TCR Ab or IL-2 into tumor-bearing mice induced TIL entrance into S phase preceding apoptosis, showing that TIL have functional TCR-mediated signal transduction in situ. Our data demonstrate that TIL, not tumor, express both Fas and FasL, are arrested in G₁, do not secrete cytokine in situ, and, upon activation in vitro and in vivo, rapidly die by activation-induced cell death.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of activation-induced cell death (AICD)³ dramatically limits peripheral T cell responses and functions primarily in elimination of mature autoreactive T lymphocytes (1). This form of tolerance is mediated through Fas-Fas ligand (L) interactions in which apoptotic cell death is induced in activated Fas⁺ autoreactive cells. Fas-mediated AICD of Ag-specific T cells has also been shown to occur in vivo after exposure to superantigen (2), oral administration of Ag (3), and virus infection (4), leading to the conclusion that AICD can function to dampen T cell immune response to a variety of Ags in addition to self Ag. Fas-mediated apoptosis is dependent on previous cell activation and requires TCR signaling (5) and cell cycling (although a CD8⁺ CTL line has been shown to be susceptible to Ag-induced TNF--mediated apoptosis in vitro when resting (6)).

CD8⁺ antitumor T cells can kill tumors in vitro, and in some cases in vivo, after either active immunization or adoptive transfer in tumor-bearing hosts (7). However, experimental immunotherapy of cancer patients, although showing significant response rates in some cases (8), is usually insufficient to completely cure established tumors. The inability of immunotherapy to cure established tumors has been recently suggested to result from impaired immune responses in patients and may be due to induction of antitumor T cell death (9). Several laboratories have hypothesized that expression of FasL by tumor cells is responsible for induction of apoptosis in tumor-reactive T cells, thereby accounting for suppression of antitumor immune response in cancer patients (10, 11, 12, 13, 14, 15, 16, 17, 18). Data in support of this notion include identification of FasL expression by human tumor cell lines and primary tumors (11), observation of apoptotic lymphocytes in cryosections prepared from primary tumors (13, 17), and induction of apoptosis in Fas⁺ Jurkat cells following coculture with tumor cell lines in vitro (13). The assertion that primary human melanoma expresses FasL has been recently refuted (19), but FasL expression by other tumor types has been reported and remains a possible basis for down-regulation of antitumor immune response (20). Because several human tumor Ags that have been identified are self Ags (e.g., p53, HER-2, MUC-1, and c-Myc) or self Ags that have acquired a single amino acid substitution compared with the corresponding wild-type sequence (e.g., Ha-ras), it is a reasonable consideration that T cells reactive to these Ags may be regulated by Fas-mediated AICD.

Based upon what is known about T cell recognition of cognate Ag, exposure of antitumor T cells to continued Ag stimulation within the tumor microenvironment is expected to result in either expansion and/or AICD in situ. In consideration of the above-mentioned concerns about the potential effects of FasL expression by tumor induction of apoptosis in T cells reactive with tumor Ags, we analyzed tumor-infiltrating lymphocytes (TIL) activation status, cytokine secretion, and proliferative response. Our data demonstrate that TIL are activated, but nonproliferative, in vitro or in situ. In addition, TIL are primed for Fas-mediated AICD but are not apoptotic in situ, as determined by TUNEL assay of tumor cryosections and flow cytometry of purified TIL after labeling with annexin V. TIL are hypersensitive to induction of AICD in vitro compared with peripheral CD8⁺ T cells, and administration of anti-TCR Ab to tumor-bearing mice induces TIL activation and AICD in situ. These results show that, although TIL are deficient in proliferative response and effector phase function, TIL TCR-mediated signal transduction is intact in situ. In addition, our results show that the Fas-mediated pathway of apoptosis is activated by TCR signaling in TIL and is dominant over survival.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C3H/HeN and C57BL/6 male mice were obtained from Jackson Laboratory (Bar Harbor, ME). Mice were housed four per cage in a barrier facility and maintained on a 12-h light, 12-h dark cycle (0700–1900 h) with ad libitum access to food and water. A sentinel program revealed that the mice were mouse hepatitis virus negative, and the tumor cell lines are mouse hepatitis virus negative as assessed by mouse assessment profile service testing. Experiments involving animals were conducted with the approval of the New York University School of Medicine Committee on Animal Research.

Tumors

The 6-1 tumor was created by expression of plasmids encoding activated murine Ha-ras plus p53 genes in primary C3H/HeN embryonic fibroblasts. The properties of this tumor have been described previously (21). MCA-38 adenocarcinoma was the gift of Y. Liu (Ohio State University, Columbus, OH). These tumors do not contain mRNA encoding FasL (data not shown). Tumor cell lines were removed from tissue culture plastic by incubation in HBSS containing 2 mM EDTA and were washed three times in HBSS. The viability of cell lines was determined by trypan blue dye exclusion, and 2 x 10⁶ cells were injected s.c. in a volume of 0.1 ml of HBSS for tumor induction.

Tissue culture

RPMI 1640 medium (BioWhittaker, Walkersville, MD) was used for isolation and culture of macrophages and T cells and was supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.002 mM L-glutamine, and 10% FBS (Intergen, Purchase, NY). DMEM was used for culture of tumor cell lines. All tissue culture supplements were supplied by Life Technologies (Grand Island, NY).

Cytofluorometry

For single-color analysis, splenocytes (10⁶), from control or tumor-bearing mice or TIL (2–3 x 10⁵) were washed once with FACS buffer (HBSS without phenol red, 1% BSA (Sigma, St. Louis, MO), and 0.1% sodium azide. Cells were incubated for 45 min on ice with 0.0005 mg of fluorochrome-conjugated Abs in a volume of 0.1 ml including 0.01 mg human IgG (Baxter Scientific Products, Chicago, IL) and 0.002 mg anti-murine CD16/32 to block nonspecific binding (clone CT-17.1; CalTag Laboratories, Burlingame, CA). After being washed with FACS buffer, cells were fixed with 1% paraformaldehyde before analysis using a FACScan Flow Cytometer (BD Biosciences, Mountain View, CA). The following Abs were used in this study: CD3 (clone 500-A2; CalTag Laboratories); CD4 (clone CT-CD4; CalTag Laboratories); CD8 (clone CT-CD8b; CalTag Laboratories); CD25 (clone PC61.5.3;CalTag Laboratories); CD28 (clone 37.51.1; CalTag Laboratories); CD40L (clone MR1; Southern Biotechnology Associates, Birmingham, AL); CD44 (clone IM7; CalTag Laboratories); CD45RB (clone 16A; CalTag Laboratories); CD45RA (clone 14.8; CalTag Laboratories); CD62L (clone MEL 14; CalTag Laboratories); CD69 (clone H1.2F3; CalTag Laboratories); CD95 (clone Jo2; BD PharMingen, San Jose, CA); CD95L (clone MFL3; BD PharMingen); CD122 (clone TM-1; BD PharMingen); F4/80 (clone CI:A3-1; CalTag Laboratories); Ly6C (clone MK1.4; Southern Biotechnology Associates); granulocytes (clone RB6-8C5; BD PharMingen); TCR- (clone H57-597; CalTag Laboratories); CTLA4 (clone VC10-4F10–11; BD PharMingen); and LFA-1 (clone 2D7; BD PharMingen).

Isolation of TIL

Tumors were dissected and chopped into small pieces using a razor blade before incubation (1 g/10 ml) with a mixture of enzymes dissolved in HBSS (collagenase type I (0.05 mg/ml), collagenase type IV (0.05 mg/ml), hyaluronidase (0.025 mg/ml), all from Sigma, DNase I (0.01 mg/ml), and soybean trypsin inhibitor (0.2 trypsin inhibitory units/ml), both from Boehringer Mannheim, Indianapolis, IN) for 15 min at 37°C. Cells were recovered by centrifugation and resuspended in a fresh aliquot of enzymes for a second 15-min incubation at 37°C. Undigested material was settled for 2 min at 0 x g, and liberated cells were recovered and washed by centrifugation in complete medium. T cells were isolated using immunomagnetic separation using type MS⁺ or VS⁺ columns and anti-CD4- or anti-CD8-conjugated magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec, Bergish-Gladbach, Germany). Briefly, 0.01 ml of bead suspension was added to 10⁷ cells in a final volume of 0.1 ml cold HBSS (containing 0.5% BSA). After incubation for 15 min on ice, cells were washed and passed through the separation column. After washing of the column with cold buffer and removal from the magnet, cells were eluted and repurified on a second column. In each experiment, aliquots of isolated cells were analyzed for cell surface expression of various markers by flow cytometry and were routinely >95% CD3⁺. T cells purified thusly from splenocytes of control mice do not express activation Ags (CD25 and CD69), do not transcribe IL-2 mRNA, and do not incorporate tritiated thymidine in proliferation assay unless stimulated in vitro.

Proliferation assay

Single-cell splenocyte suspensions were prepared by grinding spleens between the ends of microscope slides, and viability was assessed by trypan blue exclusion. Splenocytes or purified T cells were plated in 96-well plates (Flow Laboratories, McClean, VA) in complete medium; plates were coated with purified anti-TCR- mAb (H57-597; 0.01 mg/ml for 60 min at 37°C). In some experiments, purified anti-CD28 Ab (CalTag Laboratories) was used at the same concentration to stimulate cells in conjunction with anti-TCR Ab. Cultures were pulsed at 48 h with 0.5 µCi [³H]thymidine (2 Ci/mM; ICN Pharmaceuticals, Costa Mesa, CA), harvested 18–24 h later using an automated cell harvester (Wallac, Gaithersburg, MD), and incorporation of radiolabel was determined by liquid scintillation counting (Microbeta 1450; Wallac). Data are expressed as mean triplicate or quadruplicate determinations ± SE.

Chromium release assay

CTL activity of TIL was determined in standard ⁵¹Cr release assays. In brief, 10⁶ target cells, cognate tumor cells, or syngeneic but non cross-reactive MC57G tumor cells were incubated with 0.2 mCi Na[⁵¹]CrO₄ in RPMI 1640 medium for 60 min at 37°C. Cells were washed twice with complete medium and transferred to round-bottom 96-well plates at 5 x 10³ cells/well. Effector cells were prepared by in vitro culture of TIL in complete RPMI 1640 medium overnight in the presence of 100 U/ml rIL-2. Cells were added to target cells at varying numbers in a final volume of 0.2 ml to give the E:T ratios as indicated in the figure legends. After a 4-h incubation at 37°C, 0.1 ml of supernatants were harvested, and released radiolabel was determined by scintillation counting. Maximal release from target cells was determined by treatment of cells with 1% Triton X-100, spontaneous release was determined from cultures of labeled target cells incubated with medium only, and the formula used for determination of specific lysis was: [(experimental release - spontaneous release)/(maximal release - spontaneous release)] x 100.

Cell cycle analysis

CD8⁺ TIL or spleen cells were isolated by magnetic immunobeading as described above. A total of 10⁶ cells were permeabilized with cold 70% ethanol (5 min at -20°C). After being washed with PBS, the cells were incubated with 50 U RNAase A (Boehringer Mannheim) for 15 min at 37°C. The cells where then stained with 0.100 mg/ml propidium iodide (PI) for a minimum of 1 h at room temperature (RT) and analyzed by flow cytometry.

RNA isolation, reverse transcription, and PCR amplification

Total cellular RNA was isolated from T cells without in vitro stimulation, used to prepare cDNA, and used to program PCR amplification as described previously (22).

Immunocytochemistry

Tumors were removed and embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA), frozen in liquid nitrogen, and stored at -80°C. Four-micron cryosections were cut, air-dried, and fixed with cold acetone for 10 min. Specimens were treated with 0.1% hydrogen peroxide, 0.1% sodium azide in PBS to block endogenous peroxidase activity, washed in PBS, stained with biotinylated anti-CD4 or -CD8 Abs (0.01 mg/ml for 1 h at RT), washed with PBS, and reacted for 40 min at RT with streptavidin ABC (avidin/biotin complex) alkaline phosphatase (Dako, Carpinteria, CA). Reactions were visualized by development with fuchsin (Dako). Following staining for T cells, slides were treated with avidin/biotin blocking reagent (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions, stained with 0.01 mg/ml biotinylated anti-F4/80 for 1 h at RT, washed with PBS, and stained with VECSTAIN Elite ABC kit (Vector Laboratories) for 40 min at RT. Reactions were visualized by development in 0.03% hydrogen peroxide, 0.03% 3,3'-diaminobenzidine (Sigma) (in 0.05 M Tris-HCl (pH 7.6)). Slides were fixed with 4% paraformaldehyde in PBS for 10 min, counterstained with 0.5% methyl green (in 0.1 M sodium acetate (pH 4.0) for 10 min), and examined.

In situ analysis of apoptosis (TUNEL assay)

Subcutaneous tumor was isolated, embedded in Tissue-Tek OCT compound (Sakura Finetek), frozen in liquid nitrogen, and stored at -80°C until sectioning. Four-micrometer cryosections were prepared using a model CM1900 cryostat (Leica, Bannockburn, IL). Slides were air-dried at RT before fixation with cold acetone for 10 min (-20°C). Specimens were reacted with 0.01 mg/ml biotinylated anti-mouse CD8 or CD4 Abs (BD PharMingen) for 1 h at RT, followed by streptavidin ABC alkaline phosphatase (Dako) for 40 min RT. Reactions were developed using a Dako fuchsin substrate-chromogen system before fixation with 4% paraformaldehyde in PBS (10 min at RT). Between each step, specimens were washed with PBS for 30 min at RT. After T cell staining, slides were analyzed by TUNEL assay using the ApopTag Plus Peroxidase In Situ Apoptosis Detection kit (Intergen). Specimens were pretreated with 10% normal sheep serum in PBS before reaction with peroxidase-conjugated sheep polyclonal anti-digoxigenin according to the manufacturer’s instructions. Slides were counterstained with 0.5% (w/v) methyl green (in 0.1 M sodium acetate (pH 4.0) for 10 min) before photography. Controls for TUNEL assay included omission of TdT enzyme before immunocytochemistry, which revealed no reaction product.

Cell labeling with 5-bromo-2'-deoxyuridine (BrdU)

Tumor-bearing or control mice were fed BrdU (0.8 mg/ml; Sigma) in drinking water. Mice were injected with either purified control hamster IgG, anti-TCR Ab, or rIL-2 at the dosages indicated in the figures. CD8⁺ TIL or spleen cells were isolated and analyzed by flow cytometry after labeling with FITC-conjugated anti-CD8 and PE-conjugated anti-BrdU (BD PharMingen) as described (23).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of immune cell tumor infiltrates and activation status

Single-cell suspensions of tumors were prepared by enzymatic digestion after excision of any necrotic portion and characterized for the nature and extent of immune cell infiltration by flow cytometry. For these analyses, we studied two tumors, a fibrosarcoma made by expression of cDNAs encoding dominant-negative p53 plus activated Ha-ras genes in primary embryonic fibroblasts termed "6-1" (21, 22), and, separately, a chemically induced adenocarcinoma, MCA-38 (24). For all experiments, data achieved with either tumor were highly similar, if not identical, and representative data is shown. Tumors that have grown for 3 wk have undetectable infiltration of neutrophils, NK cells, or B cells. Tumors at this stage are infiltrated with low levels of both CD4⁺ and CD8⁺ T cells at approximately the same level (1–2%). In contrast to the low level of T cell infiltration, F4/80⁺ macrophages are comparatively abundant, comprising 25% of tumors at 3 wk of growth. The number of infiltrating macrophages also increases as a function of tumor size and reaches 40% of tumors at 4–5 wk of growth (2.5 cm²).

To visualize the distribution of T cells and macrophages within the tumor bed, we performed immunocytochemistry on frozen, thin sections of tumor. Relatively rare T cells were found dispersed throughout the tumor bed in apposition with both macrophages and tumor. Clusters of T cells were almost never seen; neither were close apposition of CD4⁺ and CD8⁺ TIL (data not shown).

CD8⁺ T cells were purified from tumor cell suspensions by magnetic immunobead isolation and characterized for expression of a variety of T cell surface markers by Ab labeling and flow cytometry (Fig. 1). Purified TIL express uniform levels of the TCR and CD3, showing that TIL are not contaminated with non-CD8⁺ T cells. The state of activation of purified TIL was similarly determined by flow cytometry and can be summarized as demonstrating a mixed phenotype characteristic of both activated and memory cells: CD11a⁺, CD25^-, CD40L^-, CD44⁺, CD45RA^low, CD45RB^high, CD62L^low, CD69⁺, CD95L⁺, CD122⁺, Ly6C^high.

View larger version (99K): [in this window] [in a new window]   FIGURE 1. Flow cytometric analysis of tumor-infiltrating immune cells. Single-cell suspensions were prepared by enzymatic digestion of tumor as described in Materials and Methods. After washing with PBS, cells were resuspended in FACS buffer and stained with direct fluorochrome-conjugated Abs as indicated. Isolated TIL were stained using direct fluorochrome-conjugated Abs reactive with the indicated T cell surface molecules. CD8+ TIL for these analyses were purified from tumor digests by magnetic immunobeading as described in Materials and Methods. All analyses of purified CD8+ TIL were stained with FITC-conjugated anti-CD8 plus the indicated PE-conjugated second reagent except for panels showing CD3 x TCR- and CD8 x CD40L staining, which are indicated by axis labels (PE-conjugated Ab on the y-axis and FITC-conjugated Ab on the the x-axis). These flow cytometric analyses show that tumor-infiltrating CD8+ T cells have an activated memory phenotype: CD44+, CD45RB+, CD62Llow, CD69+, CD95L+, CD122+, LFA-1+.

View larger version (47K): [in this window] [in a new window]   FIGURE 2. Tumor-infiltrating CD8+ T Cells contain mRNA encoding IL-2 and IFN-. RNA was prepared from CD4+ and CD8+ TIL or splenocytes, which had been purified by magnetic immunobeading. RNAs were used to program RT-PCRs, which were analyzed by Southern blotting (IL-2 and IFN-) as described in Materials and Methods. Positive control RNA samples were prepared by stimulating spleen-derived T cells with anti-TCR Ab for 48 h before isolation of RNA. This analysis shows that TIL contain mRNA-encoding molecules whose transcription is initiated by TCR recognition of cognate Ag.

The proliferative capacity of purified TIL was assessed by measurement of tritiated thymidine incorporation after stimulation with plate-bound anti-TCR Ab. Control CD4⁺ and CD8⁺ T cells were purified and tested for proliferation under identical conditions as for TIL (isolation from spleens using enzymatic digestion and magnetic immunobeading). Control T cells and TIL do not incorporate tritiated thymidine when nonimmune hamster IgG is used instead of anti-TCR Ab, and both CD4⁺ and CD8⁺ control T cells incorporate thymidine upon activation in vitro (Fig. 3A). However, incorporation of thymidine into both CD4⁺ and CD8⁺ TIL is dramatically reduced compared with an equivalent number of control T cells.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. A, Proliferation assay of TIL. TIL or control spleen T cells were isolated by magnetic immunobeading as described in Materials and Methods. Proliferation of purified T cells was tested by incubation in 96-well plates (5 x 104 cells/well) that were precoated with saturating amounts of anti-TCR or control Ab. Wells were pulsed with tritiated thymidine at 48 h, harvested at 72 h, and incorporated radiolabel was detected by scintillation counting. These experiments show that both CD4+ and CD8+ TIL are inhibited in proliferation in comparison to control spleen T cells. B, CD8+ TIL have Ag-specific lytic function. Cytolytic activity of MCA-38 CD8+ TIL cultured overnight in the presence of 100 U/ml exogenous rIL-2 was determined in chromium release assays with autologous MCA-38 tumor cells () or syngeneic but non-cross-reactive MC57G tumor cells (•) as target cells. C, IFN- release by TIL is stimulated by primary cognate tumor. CD8+ TIL were prepared from MCA-38 tumors by positive selection with magnetic immunobeads. CD8+ T cell-depleted digests of primary tumors were further depleted of CD4+ T cells and macrophages by negative selection using appropriate magnetic immunobeads and were used immediately to stimulate cognate TIL for analysis of IFN- secretion in vitro. Syngeneic MC57G tumor cells were used in a similar manner to stimulate anti-MCA-38 TIL in vitro. IFN- ELISA was performed as described in the legend to Fig. 4.

Visualization of TIL cultures during the proliferation assay by microscopy showed that, in comparison to control T cells, TIL did not blast and appeared greatly reduced in number (data not shown). This was surprising because cell surface activation Ags were strongly expressed by TIL (Fig. 1), and we anticipated that TIL would vigorously proliferate. Therefore, we considered the possibility that TIL were dying upon activation in vitro. To directly assess this possibility, TIL were isolated and stimulated with plate-bound anti-TCR Ab for increasing time and cell recovery, determined by enumeration of viable cells after staining with PI (Fig. 4A). (CD4⁺ TIL behaved identically with CD8⁺ TIL in subsequent experiments; for simplicity, only CD8⁺ TIL data is shown). The number of control T cells increased dramatically under these activating conditions. In contrast, the number of TIL activated with anti-TCR Ab rapidly declines such that, compared with control T cells, by 72 h, <10% of cells are present. The kinetics of the decrease in cell recovery suggests that the basis for reduced incorporation of tritiated thymidine into TIL activated in vitro is the dramatic reduction in cell number.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. A, Recovery of CD8+ TIL after activation in vitro. CD8+ TIL or control spleen CD8+ T cells were isolated by magnetic immunobeading as described in Materials and Methods. Cells were plated in triplicate in 96-well plates in the presence of saturating amounts of plate-bound anti-TCR Ab (0.1 ml at 0.01 mg/ml). Wells were harvested at the times indicated, and viable cells were enumerated by staining with PI and flow cytometry. This analysis shows that recovery of viable CD8+ TIL decreases dramatically following ligation of the TCR in vitro. B, CD8+ TIL become annexin V+ after TCR ligation in vitro. TIL or control spleen CD8+ T cells were isolated by magnetic immunobeading as described in Materials and Methods. Cells were plated in 24-well plates in the presence of saturating amounts of plate-bound anti-TCR Ab (0.1 ml at 0.01 mg/ml). Wells were harvested at the times indicated and analyzed by flow cytometry after staining with FITC-conjugated annexin V. This analysis shows that freshly isolated TIL do not bind annexin V but express cell surface phosphatidylserine after activation in vitro. C, TIL cytokine ELISA. TIL were isolated, plated in vitro in the presence or absence of plate-bound anti-TCR Ab for the indicated times (2 x 105 cells/well), and conditioned medium was collected and analyzed by ELISA for the presence of IL-2 or IFN- as described (40 ). Standard curves made using recombinant cytokines were linear to 50 pg/ml. D, Flow cytometric analysis of IL-2 and IFN- expression. TIL were isolated and incubated in medium containing 3 µM monensin for 5 h with or without activation with plate-bound anti-TCR Ab before intracellular cytokine staining with fluorochrome-conjugated anti-IL-2 or IFN-. This experiment shows that TIL do not express IL-2 or IFN- unless activated, and only a small percentage of TIL are secreting. E, Proliferative deficit of CD8+ TIL after TCR ligation in vitro is reversed by blocking Fas-FasL interaction. TIL were isolated by magnetic immunobeading as described in Materials and Methods. Proliferation of purified T cells was determined by incubation in 96-well plates (5 x 104 cells/well) that were precoated with saturating amounts of anti-TCR or control Ab (not stimulated). Wells contained either control of blocking Ab to FasL (0.01 mg/ml) as indicated. Wells were pulsed with tritiated thymidine at 48 h and were harvested at 72 h. Incorporated radiolabel was detected by scintillation counting. This experiment shows that incorporation of thymidine into activated CD8+ TIL is restored if Fas-FasL interaction is blocked.

The phenotype of TIL after activation was further examined by analysis of cytokine production in vitro. TIL were isolated, activated with anti-TCR Ab, and the levels of IL-2 and IFN- secreted were determined by ELISA of conditioned medium (Fig. 4C). Freshly isolated TIL do not secrete detectable cytokine in the absence of stimulation, although mRNA encoding both IL-2 and IFN- is present. Secretion of IFN- was detected after 4 h in vitro and, by 6 h, the level produced was substantial (2000 U/10⁶ cells in 6 h) and almost the same as that secreted by activated primary MLR CD8⁺ T cells (data not shown). Control spleen CD8⁺ T cells activated identically do not produce detectable IL-2 or IFN- (data not shown). In addition, corroborating the ELISA findings, intracellular flow cytometric analysis of freshly isolated TIL did not detect the presence of cytokine proteins (Fig. 4D). Intracellular staining after TIL isolation and activation in the presence of monensin showed that a portion of TIL secrete cytokines after activation in vitro; without activation there was no detectable cytokine protein. This analysis shows that, upon activation, substantial TIL cytokine synthesis and secretion occurs preceding AICD.

Expression of cell surface phosphatidylserine after TIL activation in vitro suggested that TIL die by induction of apoptosis. Because freshly isolated TIL express both cell surface Fas and FasL and become apoptotic upon activation in vitro, TIL appear to die by activation-induced cell death. The role of Fas and FasL in this process was examined by inclusion of anti-FasL Ab (Fig. 4E) in the in vitro proliferation assay. Inclusion of isotype-matched control Ab had no effect on thymidine incorporation into CD8⁺ TIL because TIL proliferation, as noted previously, was depressed relative to control spleen-derived T cells. In contrast, inclusion of anti-FasL Ab restored proliferation close to the level of control T cells. Anti-FasL Ab also restored recovery of TIL to levels seen with control T cells (data not shown). Collectively, the experiments shown in Fig. 4 show that TIL are primed for AICD upon in vitro ligation of the TCR.

TIL cell cycle status

TIL cell cycle status was assessed by flow cytometry after staining with PI. Despite expression of a variety of cell surface activation markers (Fig. 1), freshly isolated TIL are not cycling because only 6–8% of cells are in S phase (compared with 4–6% of control spleen T cells; data not shown). Upon stimulation with anti-TCR Ab in vitro for increasing time, the same percentage of TIL enter S phase as do control T cells; within 24 h of activation, 20% of cells are in S phase, and the percentage of cells in S phase increases to >50% at 48 h (data not shown). The number of TIL available for cell cycle analysis at any given time after activation in vitro decreases dramatically (Fig. 4A), but, of those cells remaining in culture, the percentage in S phase is equivalent to that of control cells. This suggests that, although TIL can be stimulated through the TCR and progress with normal kinetics into S phase, the Fas-mediated pathway of apoptosis is activated and dominant over survival.

TIL are not apoptotic in situ

Several laboratories have reported that expression of FasL by tumors induces death of Fas⁺ antitumor T cells, thereby contributing to tumor escape from immune destruction (20). This notion has been called into question by the rigorous demonstration that human melanoma do not express FasL (19); however, the possibility that other tumor types may express FasL that contributes to antitumor T cell inactivation remains open. In addition, because TIL express both Fas and FasL, the possibility exists that TIL may die upon contact in vitro by fratricide or suicide. In consideration of this point, viable TIL that do not express cell surface phosphatidylserine can be isolated, strongly implying that TIL are not apoptotic. However, we considered that apoptotic TIL may be rapidly cleared from the tumor and may therefore be unable to be recovered by magnetic immunobeading, a possibility enhanced by the abundance of macrophage in tumors. To directly assess whether TIL are apoptotic, we performed in situ immunohistological characterization of apoptosis in tumors. Tumor cryosections were analyzed by TUNEL assay and immunocytochemical labeling for either CD4 or CD8. We found that, with the exception of the necrotic portion of the tumor, there is very little apoptosis of any cell type in the tumor bed and that only 3–8% of TIL were TUNEL⁺ (data not shown). TUNEL⁺ TIL were in the early phase of apoptosis by several criteria: cells had intact plasma membrane with no apparent surface blebs, HRP immunocytochemical reaction product was localized only to nuclei, and TIL were not condensed (i.e., they contained abundant cytoplasm that was unstained). We analyzed multiple individual tumors at different stages of growth and viewed over 2200 individual microscopic fields in this determination (summarized in Table I).

TIL have an activated phenotype in situ, evidenced by expression of cell surface activation markers and stable cytokine RNA but are not cycling nor apoptotic. However, upon activation in vitro, TIL are readily induced to AICD. Therefore, we considered the possibility that TIL in situ are not apoptotic because they receive suboptimal activation stimuli that is insufficient to drive them out of G₁ phase and into subsequent apoptosis. Alternatively, the activation threshold may be higher for TIL than for control cells. These possibilities were tested in the next sets of experiments.

We first asked whether purified TIL would proliferate under suboptimal activation conditions in vitro. Control T cells and purified TIL were stimulated with increasing amounts of anti-TCR Ab before measurement of incorporation of tritiated thymidine. We found that, under suboptimal activation conditions, TIL incorporate identical levels of thymidine as control T cells (Fig. 5A). At the lower concentrations of anti-TCR Ab, there was a dose-dependent increase in thymidine incorporation into TIL whose magnitude was identical with control T cells. However, as has been noted above, at saturating anti-TCR Ab concentration (10 µg/ml), incorporation of thymidine into TIL is dramatically depressed relative to control T cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. TIL analysis under suboptimal activating stimuli in vitro. A, Thymidine incorporation. TIL or control spleen T cells were isolated by magnetic immunobeading as described in Materials and Methods. Proliferation of purified T cells was tested by incubation in 96-well plates (5 x 104 cells/well) that were precoated with different concentrations of anti-TCR Ab as indicated. Cultures were pulsed with tritiated thymidine at 48 h, wells were harvested at 72 h, and incorporated radiolabel was detected by scintillation counting. This experiment shows that CD8+ TIL incorporate tritiated thymidine to the same extent as normal spleen T cells at suboptimal activation conditions but do not incorporate thymidine at saturating conditions. B, Recovery of CD8+ TIL after activation under suboptimal activating stimuli in vitro. CD8+ TIL were isolated by magnetic immunobeading as described in Materials and Methods. A total of 105 cells were plated in triplicate in 96-well plates that were precoated with increasing concentrations of plate-bound anti-TCR Ab. Wells were harvested at the times indicated, and viable cells were enumerated by staining with PI and flow cytometry. This analysis shows that recovery of viable CD8+ TIL is dramatically reduced at late times after in vitro activation at any concentration of stimulating Ab. At early times (12 h), recovery is inversely proportional to the level of activation in vitro. C, Cell cycle analysis of CD8+ TIL after activation under suboptimal activating stimuli in vitro. TIL or control spleen CD8+ T cells were isolated by magnetic immunobeading as described in Materials and Methods. Cells were plated in 24-well plates that were precoated with increasing concentrations of plate-bound anti-TCR Ab as indicated. Wells were harvested at 24 h, and cell cycle status was determined by flow cytometry following incubation with PI. This analysis shows that TIL enter S phase after activation in vitro at suboptimal activating conditions in vitro. D, CD8+ TIL become annexin V+ after TCR ligation under suboptimal activating stimuli in vitro. TIL or control spleen CD8+ T cells were isolated by magnetic immunobeading as described in Materials and Methods. Cells were incubated in vitro in plates that were precoated with increasing concentrations of plate-bound anti-TCR Ab as indicated. Wells were harvested at 24 h and analyzed by flow cytometry after staining with FITC-conjugated annexin V. This analysis shows that TIL express cell surface phosphatidylserine to a greater extent compared with control T cells after activation in vitro at suboptimal activation conditions. In addition, expression of cell surface phosphatidylserine by TIL occurs more rapidly than control T cells at saturating conditions.

Suboptimal stimulation in vitro resulted in tritiated thymidine incorporation into TIL at the level of control T cells and also decreased recovery of TIL at later time points, suggesting that quiescent TIL were highly sensitive to TCR ligation resulting in AICD in vitro. We measured the effect of suboptimal activation upon cell cycle progression (Fig. 5C). After 24 h of activation using the lowest concentration of activating Ab (0.1 µg/ml anti-TCR), control T cells remain in G₁ and, at 1 µg/ml, control T cells are slightly stimulated to enter S phase. In contrast, a high percentage of TIL enter S phase at the lowest concentration of stimulating Ab used, and the number of cycling cells increases in a dose-dependent manner. This experiment showed that TIL rapidly enter S phase under conditions of TCR ligation wherein control T cells are unaffected and remain in G₁.

Induction of apoptosis at suboptimal conditions of TCR ligation was examined because the recovery of TIL is dramatically reduced under these conditions of activation in vitro (Fig. 5D). Control T cells, although slightly induced to incorporate thymidine and enter S phase under suboptimal conditions, do not become apoptotic. In contrast, TIL cell surface phosphatidylserine expression is high after stimulation with the lowest anti-TCR Ab concentration tested, and, at the intermediate concentration, cells are highly apoptotic. Collectively, the experiments shown in Fig. 5 suggest that, relative to control T cells, TIL are hypersensitive to TCR ligation and enter S phase before becoming apoptotic.

Injection of tumor-bearing mice with anti-TCR Ab or rIL-2 activates TIL in situ and induces TIL apoptosis

Activation of purified T cells by TCR ligation in vitro is widely accepted to reflect Ag recognition in terms of activation of signal transduction. However, we considered the possibility that, within the tumor microenvironment, the presence of tumor cells, stroma, or infiltrating macrophage may alter TIL TCR responsiveness. Therefore, we asked whether ligation of TIL TCR in situ could induce either TIL activation or apoptosis. Tumor-bearing mice were injected with anti-TCR Ab, control hamster IgG, or rIL-2. Twelve hours later, TIL were isolated and plated in the presence of radiolabeled thymidine to measure DNA synthesis (Fig. 6A). TIL isolated from mice injected with anti-TCR Ab incorporated thymidine without further stimulation in vitro, whereas if mice were injected with control IgG, little proliferation was seen. Spleen CD8⁺ T cells were also activated by injection of anti-TCR Ab, showing that activation of T cells in vivo by anti-TCR Ab was not restricted to TIL. As was seen for injection of anti-TCR Ab, TIL are activated by IL-2 administration such that they incorporate thymidine in vitro without additional activation in vitro. In contrast to activation by injection of anti-TCR Ab, spleen T cells from the same tumor-bearing mice are not activated by IL-2, suggesting that TIL are primed in situ.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Injection of tumor-bearing mice with anti-TCR Ab activates TIL. Tumor-bearing mice were injected i.p. with either anti-TCR IgG at the doses indicated or with rIL-2 (2000 U/mouse). Tumor-bearing mice were separately injected with control hamster IgG (0.01 or 0.05 mg/injection; control), and non-tumor-bearing mice were injected with either anti-TCR, control hamster IgG, or rIL-2 before isolation of CD8+ spleen cells and TIL by magnetic immunobeading. A, Activation of TIL in situ induces proliferation. Tumor-bearing mice were injected i.v. with 0.01 mg anti-TCR, 0.05 mg control IgG, or rIL-2 (2000 U/mouse). Twelve hours later, cells were isolated and plated at 5 x 104 cells/well in triplicate in 96-well plates. Wells were pulsed with tritiated thymidine at the time of cell plating and were harvested for scintillation counting 16 h later. This experiment shows that TIL are activated in situ by injection of either anti-TCR Ab or IL-2 such that, after isolation, they incorporate tritiated thymidine in vitro without further activation in vitro. B, Activation in situ induces increased BrdU labeling of TIL. Tumor-bearing mice were fed BrdU in water at the same time as injection with 0.01 mg anti-TCR or control IgG. Twenty hours later, TIL were isolated and stained with PE-conjugated isotype-matched control or PE-conjugated anti-BrdU Ab before analysis by flow cytometry. This experiment shows that TIL are activated by injection of anti-TCR Ab such that they incorporate BrdU in situ to a greater extent than mice injected with control IgG.

The previous two experiments are interpreted to mean that TIL are able to be activated in situ to enter S phase. However, we have also shown that TIL are hypersensitive to AICD in vitro. Therefore, we asked whether activation in situ induces AICD by analyzing TIL for annexin V reactivity after activation in situ (Fig. 7). Tumor-bearing mice were injected with control IgG, anti-TCR Ab, or rIL-2 before isolation and flow cytometric analysis after annexin V labeling. Control IgG injection induced modest TIL phosphatidylserine expression in TIL and spleen T cells of tumor-bearing mice but not in the control non-tumor-bearing mice (9% of TIL vs 3% of control spleen CD8⁺ T cells). In contrast, after anti-TCR Ab injection into tumor-bearing mice, significant phosphatidylserine expression was induced in both TIL and, to a lesser extent, spleen T cells. In addition, administration of rIL-2 to tumor-bearing mice caused a rapid increase in TIL annexin V reactivity. Contrary to induction of proliferation in spleen T cells in mice receiving anti-TCR Ab, significant AICD was not induced in spleen T cells after IL-2 injection. The susceptibility of TIL to IL-2-induced AICD suggests that, in accordance with the data of Refaeli and colleagues (who showed that repeatedly activated T cells, but not nonactivated T cells, are susceptible to IL-2-induced AICD (1)), TIL are previously activated.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Activation in situ induces increased annexin V labeling of TIL. Tumor-bearing mice were injected with anti-TCR or control IgG as indicated. Twelve hours later, TIL or spleen cells were isolated, stained with FITC-conjugated annexin V, and analyzed by flow cytometry. This experiment shows that, in comparison to CD8+ splenocytes, TIL are primed for rapid induction of apoptosis in vivo.

This experiment illustrates two additional important points. First, the fact that annexin V⁺ TIL can be isolated in high numbers shows that apoptotic TIL are isolable from tumor digests, and, therefore, previous experiments that quantified apoptotic TIL were not likely to reflect false-negative results due to a potential inability to recover apoptotic TIL. Second, because the level of TUNEL⁺ macrophages in tumor cryosections is very low and the TUNEL reaction detects labeling of apoptotic T cell DNA after phagocytosis of T cells by macrophage in vitro (data not shown), annexin V⁺ apoptotic TIL are not immediately phagocytotosed in situ, which would also lead to underestimation of the extent of TIL apoptosis in situ.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Apoptosis of infiltrating T cells in several human cancers has been reported for several tumor types (17, 25, 26, 27, 28), and FasL expression by tumor cells has been postulated to be the basis for TIL apoptosis because of the observations that primary tumor cells can express FasL (20). However, tumor expression of FasL has recently been refuted for human melanoma (29). Therefore, the conclusion that tumor cell expression of FasL is the basis for the enhanced apoptosis of TIL remains unsettled. Irrespective of whether tumors express FasL, some TIL are undoubtedly apoptotic, although the extent of TIL apoptosis is variable and likely depends upon multiple factors including extent of necrosis and tumor stage and size (30). In addition, because Ag recognition by T cells can result in AICD, an unknown proportion of TIL apoptosis may be due to recognition of tumor Ag on tumor cells (31). However, our data call into question the notion that TIL apoptosis is extensive and, consequently, whether TIL apoptosis is a significant mechanism of tumor escape from immune response (13, 17, 20, 26, 32). We find that there is a low level of apoptotic TIL in situ. The finding of only modest levels of TIL apoptosis may reflect an inherent difference between murine and human tumors, although previous reports that examined TIL apoptosis in human tumors neither quantitated the level of apoptotic T cells by combination of immunocytochemistry and TUNEL assay, nor did they specifically exclude analysis of necrotic tumor (17, 26).

Although TIL express cell surface activation Ags and contain cytokine mRNAs indicative of activation, TIL are not actively proliferating or engaged in effector phase function in situ. This conclusion is reached by several observations. First, antitumor T cells do not incorporate BrdU after arrival into tumor tissue. This contention is supported by the findings that, immediately upon isolation, a low percentage of TIL are in S phase, and isolated TIL do not incorporate tritiated thymidine in vitro. Furthermore, although TIL secrete high levels of IFN- upon activation in vitro, cytokine secretion in situ is not detected, implying that they are incompletely activated. Finally, the very low level of apoptosis of tumor cells (as revealed by TUNEL assay of tumor cryosections) demonstrates that TIL are nonlytic in situ. This conclusion is further strengthened by our observation that freshly purified TIL are nonlytic in vitro (data not shown).

TIL are nonfunctional in situ but are Ag-specific. This conclusion is based upon our observation that, in addition to not proliferating and secreting cytokine in situ as shown above and although TIL are not lytic upon isolation, after purification and a short period of recovery in in vitro culture, tumor-specific lytic function is recovered. Freshly isolated TIL secrete IFN- upon stimulation with cognate tumor, showing that effector phase functions of TIL are differentially inhibited. This contention is also supported by the work of others using transgenic murine models expressing TCR that recognize cognate tumor Ags wherein TIL are shown to accumulate in tumor tissue but are nonlytic in situ or in vitro (33). In addition, as shown above, TIL are partially activated in situ (expression of cytokine mRNAs) and express cell surface markers characteristic of memory/effector T cells. Collectively, our data show that, instead of actively responding to activation by cognate tumor Ag in situ, TIL are quiescent and nonresponsive.

There have been many reports of use of TIL in antitumor adoptive transfer experiments both in rodent models (34) and experimental human immunotherapy (35). For that purpose, TIL are isolated and cultured in the presence of high concentrations of IL-2 to expand sufficiently for therapy. Our data is consistent with those publications. If TIL are purified and cultured briefly in the presence of IL-2, they enter the cell cycle and regain tumor-specific lytic function. Tumor-draining lymph node (LN) cells are also used in adoptive immunotherapy protocols (36). LN T cells have a different phenotype in terms of responses to activation with anti-TCR Ab, demonstrating a fundamental difference compared with TIL: LN cells are not primed for AICD, nor are they apoptotic in vivo. We believe that the observed differences reflect the influence of the tumor microenvironment on T cells in situ.

With regard to potential similarity of TIL to memory T cells, TIL express many cell surface markers that are characteristic of memory cells, except TIL are CD69^high, whereas memory cells are CD69^low. In addition, TIL share one property that is characteristic of CD8⁺ memory cells as defined in two recent studies: rapid secretion of IFN- upon TCR ligation in vitro (37, 38). Furthermore, TIL share another characteristic of memory T cells in that the kinetics of activation and the activation threshold are lower than naive T cells; memory T cells rapidly proliferate under suboptimal activating conditions that do not activate naive T cells, although this point is controversial. In contrast to memory cells, TIL die by apoptosis under weaker activation conditions compared with control or memory T cells. Another difference between TIL and memory CD8⁺ T cells is the ability to induce CTL activity; memory CTL require 24 h of ex vivo activation to develop lytic activity (37), whereas, as is seen upon assay of proliferative capacity, when stimulated by TCR ligation in vitro, TIL are nonlytic and undergo apoptotic death. Therefore, although TIL have cell surface markers of memory cells, in distinction to memory cells, TIL are nonproliferative and are primed for AICD.

TIL AICD in vitro is mediated by Fas-FasL interaction, as shown by inhibition of cell death upon inclusion of blocking anti-FasL Ab. Fas-mediated TIL death may occur in situ, as suggested by the observation that TIL appear as isolated cells within tumor. Based upon our in vitro studies, if TIL were closely apposed in situ, Fas-mediated AICD may occur and these T cells would be eliminated. However, this contention may be a peculiarity of the murine tumors studied because of two considerations. First, tumor growth in transgenic mice expressing TCR reactive with cognate tumor Ag accumulate significant TIL populations that are also not apoptotic in situ (S. Radoja, unpublished observation). Second, human tumors frequently have high levels of infiltrating nonapoptotic T cells (39). Therefore, we consider the observation that FasL⁺ TIL are not apoptotic in situ to have an unknown basis. Perhaps, because AICD requires TCR-mediated signal transduction in addition to Fas-FasL interaction, it is possible that TCR-mediated signal transduction is blocked in TIL such that the requisite TCR signal is unable to be transmitted, thereby preventing AICD. Another consideration is that TIL TCR-mediated signal transduction is intact, but cognate Ag expression by tumor cells in situ is deficient such that TIL are not being stimulated by Ag recognition. Our data wherein TIL were activated in situ in tumor-bearing mice, resulting in increased TIL apoptosis, supports this contention. These two possibilities are currently being further tested.

The biochemical basis for induction of TIL unresponsiveness within the tumor is not yet understood but has several consequences. On one hand, being nonresponsive, TIL cannot kill antigenic tumors, which confers a growth advantage to tumors. On the other hand, if, upon recognition of cognate tumor Ag, TIL were fully responsive, induction of AICD would likely result and TIL would then be eliminated by apoptosis. There are several reports of apoptotic T cells in primary human tumors, although an accurate assessment of the extent of AICD in situ remains to be determined. Therefore, understanding the factors that influence induction of TIL nonresponsiveness in situ, which includes defining how some TIL can avoid induction of AICD and maintain effector phase functions, is a major objective for tumor immunology.

	Acknowledgments

 
We thank John Hirst for performing the flow cytometry analysis, Pam Cowin for use of the microtome, and Stanislav Vukmanovic for editorial comments.

	Footnotes

 
¹ Flow cytometric facilities were supported in part by National Institutes of Health Grant CA 16087 to the Kaplan Cancer Center.

² Address correspondence and reprint requests to Dr. Alan B. Frey, Department of Cell Biology, New York University School of Medicine, Room MSB 690, 550 First Avenue, New York, NY 10016.

³ Abbreviations used in this paper: AICD, activation-induced cell death; L, ligand; LN, lymph node; PI, propidium iodide; TIL, tumor infiltrating lymphocytes; RT, room temperature; BrdU, 5-bromo-2'-deoxyuridine.

Received for publication December 26, 2000. Accepted for publication March 12, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Refaeli, Y., L. Van Parijs, C. A. London, J. Tschopp, A. K. Abbas. 1998. Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis. Immunity. 8:615.[Medline]
2. McCormack, J. E., J. E. Callahan, J. Kappler, P. C. Marrack. 1993. Profound deletion of mature T cells in vivo by chronic exposure to exogenous superantigen. J. Immunol. 150:3785.[Abstract]
3. Chen, Y., J. Inobe, R. Marks, P. Gonnella, V. K. Kuchroo, H. L. Weiner. 1995. Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature 376:177.[Medline]
4. Moskophidis, D., F. Lechner, H. Pircher, R. M. Zinkernagel. 1993. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector T cells. Nature 362:758.[Medline]
5. Suzuki, G., Y. Kawase, S. Koyasu, I. Yahara, Y. Kobayashi, R. H. Schwartz. 1988. Antigen-induced suppression of the proliferative response of T cell clones. J. Immunol. 140:1359.[Abstract]
6. Alexander-Miller, M. A., G. R. Leggatt, A. Sarin, J. A. Berzofsky. 1996. Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL. J. Exp. Med. 184:485.[Abstract/Free Full Text]
7. Evans, R., S. J. Kamdar, T. M. Duffy, D. M. Krupke, J. A. Fuller, M. E. Dudley. 1995. The therapeutic efficacy of murine anti-tumor T cells: freshly isolated T cells are more therapeutic than T cells expanded in vitro. Anticancer Res. 15:441.[Medline]
8. Rosenberg, S. A.. 1998. New opportunities for the development of cancer immunotherapies. Cancer J. Sci. Am. 4:(Suppl. 1):S1.
9. Finke, J., S. Ferrone, A. Frey, A. Mufson, A. Ochoa. 1999. Where have all the T cells gone: mechanisms of immune evasion by tumors. Immunol. Today 20:158.[Medline]
10. Bennett, M. W., J. O’Connell, G. C. O’Sullivan, C. Brady, D. Roche, J. K. Collins, F. Shanahan. 1998. The Fas counterattack in vivo: apoptotic depletion of tumor-infiltrating lymphocytes associated with Fas ligand expression by human esophageal carcinoma. J. Immunol. 160:5669.[Abstract/Free Full Text]
11. Hahne, M., D. Rimoldi, M. Schroter, P. Romero, M. Schreier, L. E. French, P. Schneider, T. Bornand, A. Fontana, D. Lienard, J. Cerottini, J. Tschopp. 1996. Melanoma cell expression of Fas (Apo-1/CD95) ligand: implications for tumor immune escape. Science 274:1363.[Abstract/Free Full Text]
12. O’Connell, J., G. C. O’Sullivan, J. K. Collins, F. Shanahan. 1996. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J. Exp. Med. 184:1075.[Abstract/Free Full Text]
13. Rabinowich, H., T. E. Reichert, Y. Kashii, B. R. Gastman, M. C. Bell, T. L. Whiteside. 1998. Lymphocyte apoptosis induced by Fas ligand- expressing ovarian carcinoma cells: implications for altered expression of T cell receptor in tumor-associated lymphocytes. J. Clin. Invest. 101:2579.[Medline]
14. Saas, P., P. R. Walker, M. Hahne, A. L. Quiquerez, V. Schnuriger, G. Perrin, L. French, E. G. Van Meir, N. de Tribolet, J. Tschopp, P. Y. Dietrich. 1997. Fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain?. J. Clin. Invest. 99:1173.[Medline]
15. Shiraki, K., N. Tsuji, T. Shioda, K. J. Isselbacher, H. Takahashi. 1997. Expression of Fas ligand in liver metastases of human colonic adenocarcinomas. Proc. Natl. Acad. Sci. USA 94:6420.[Abstract/Free Full Text]
16. Strand, S., W. J. Hofmann, H. Hug, M. Muller, G. Otto, D. Strand, S. M. Mariani, W. Stremmel, P. H. Krammer, P. R. Galle. 1996. Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells–a mechanism of immune evasion?. Nat. Med. 2:1361.[Medline]
17. Uzzo, R. G., P. Rayman, V. Kolenko, P. E. Clark, T. Bloom, A. M. Ward, L. Molto, C. Tannenbaum, L. J. Worford, R. Bukowski, et al 1999. Mechanisms of apoptosis in T cells from patients with renal cell carcinoma. Clin. Cancer Res. 5:1219.[Abstract/Free Full Text]
18. Walker, P. R., P. Saas, P. Y. Dietrich. 1997. Role of Fas ligand (CD95L) in immune escape: the tumor cell strikes back. J. Immunol. 158:4521.[Abstract]
19. Chappell, D. B., T. Z. Zaks, S. A. Rosenberg, N. P. Restifo. 1999. Human melanoma cells do not express Fas (Apo-1/CD95) ligand. Cancer Res. 59:59.[Abstract/Free Full Text]
20. O’Connell, J., M. W. Bennett, G. C. O’Sullivan, J. K. Collins, F. Shanahan. 1999. The Fas counterattack: cancer as a site of immune privilege. Immunol. Today 20:46.[Medline]
21. Appleman, L. J., J. Uyeki, A. B. Frey. 1995. Mouse embryo fibroblasts transformed by activated ras or dominant-negative p53 express cross-reactive tumor rejection antigens. Int. J. Cancer 61:887.[Medline]
22. Radoja, S., T. D. Rao, D. Hillman, A. B. Frey. 2000. Mice bearing late-stage tumors have normal functional systemic T cell responses in vitro and in vivo. J. Immunol. 164:2619.[Abstract/Free Full Text]
23. Hurst, S. D., C. J. Cooper, S. M. Sitterding, J. Choi, R. L. Jump, A. D. Levine, T. A. Barrett. 1999. The differentiated state of intestinal lamina propria CD4⁺ T cells results in altered cytokine production, activation threshold, and costimulatory requirements. J. Immunol. 163:5937.[Abstract/Free Full Text]
24. Tan, M. H., E. D. Holyoke, M. H. Goldrosen. 1976. Murine colon adenocarcinomas: methods for selective culture in vitro. J. Natl. Cancer Inst. 56:871.
25. Eerola, A. K., Y. Soini, P. Paakko. 1999. Tumour infiltrating lymphocytes in relation to tumour angiogenesis, apoptosis and prognosis in patients with large cell lung carcinoma. Lung Cancer 26:73.[Medline]
26. Gastman, B. R., Y. Atarshi, T. E. Reichert, T. Saito, L. Balkir, H. Rabinowich, T. L. Whiteside. 1999. Fas ligand is expressed on human squamous cell carcinomas of the head and neck, and it promotes apoptosis of T lymphocytes. Cancer Res. 59:5356.[Abstract/Free Full Text]
27. Munakata, S., T. Enomoto, M. Tsujimoto, Y. Otsuki, H. Miwa, H. Kanno, K. Aozasa. 2000. Expressions of Fas ligand and other apoptosis-related genes and their prognostic significance in epithelial ovarian neoplasms. Br. J. Cancer 82:1446.[Medline]
28. O’Connell, J., M. W. Bennett, G. C. O’Sullivan, J. O’Callaghan, J. K. Collins, F. Shanahan. 1999. Expression of Fas (CD95/APO-1) ligand by human breast cancers: significance for tumor immune privilege. Clin. Diagn. Lab. Immunol. 6:457.[Abstract/Free Full Text]
29. Restifo, N. P.. 2000. Not so fas: re-evaluating the mechanisms of immune privilege and tumor escape. Nat. Med. 6:493.[Medline]
30. Whiteside, T. L.. 1998. Immune cells in the tumor microenvironment: mechanisms responsible for functional and signaling defects. Adv. Exp. Med. Biol. 451:167.[Medline]
31. Levey, D., P. Srivastava. 1996. Alterations in T cells of cancer-bearers: whence specificity?. Immunol. Today 17:365.[Medline]
32. Gastman, B. R., D. E. Johnson, T. L. Whiteside, H. Rabinowich. 2000. Tumor-induced apoptosis of T lymphocytes: elucidation of intracellular apoptotic events. Blood 95:2015.[Abstract/Free Full Text]
33. Prevost-Blondel, A., C. Zimmermann, C. Stemmer, P. Kulmburg, F. M. Rosenthal, H. Pircher. 1998. Tumor-infiltrating lymphocytes exhibiting high ex vivo cytolytic activity fail to prevent murine melanoma tumor growth in vivo. J. Immunol. 161:2187.[Abstract/Free Full Text]
34. Maeda, K., R. Lafreniere, L. M. Jerry. 1991. Production and characterization of tumor infiltrating lymphocyte clones derived from B16–F10 murine melanoma. J. Invest. Dermatol. 97:183.[Medline]
35. Topalian, S. L., L. M. Muul, D. Solomon, S. A. Rosenberg. 1987. Expansion of human tumor infiltrating lymphocytes for use in immunotherapy trials. J. Immunol. Methods 102:127.[Medline]
36. Plautz, G. E., J. E. Touhalisky, S. Shu. 1997. Treatment of murine gliomas by adoptive transfer of ex vivo activated tumor-draining lymph node cells. Cell. Immunol. 178:101.[Medline]
37. Bachmann, M. F., M. Barner, A. Viola, M. Kopf. 1999. Distinct kinetics of cytokine production and cytolysis in effector and memory T cells after viral infection. Eur. J. Immunol. 29:291.[Medline]
38. Zimmermann, C., A. Prevost-Blondel, C. Blaser, H. Pircher. 1999. Kinetics of the response of naive and memory CD8 T cells to antigen: similarities and differences. Eur. J. Immunol. 29:284.[Medline]
39. Lopez, C., T. D. Rao, H. Feiner, R. Shapiro, J. Marks, A. B. Frey. 1998. Repression of interleukin-2 mRNA translation in primary human breast carcinoma tumor infiltrating lymphocytes. Cell. Immunol. 190:141.[Medline]
40. Frey, A. B., T. D. Rao. 1999. NKT cell cytokine imbalance in murine diabetes mellitus. Autoimmunity 29:201.[Medline]

This article has been cited by other articles:

				M. Aubert, M. Yoon, D. D. Sloan, P. G. Spear, and K. R. Jerome The Virological Synapse Facilitates Herpes Simplex Virus Entry into T Cells J. Virol., June 15, 2009; 83(12): 6171 - 6183. [Abstract] [Full Text] [PDF]

				R. Mortarini, C. Vegetti, A. Molla, F. Arienti, F. Ravagnani, A. Maurichi, R. Patuzzo, M. Santinami, and A. Anichini Impaired STAT Phosphorylation in T Cells from Melanoma Patients in Response to IL-2: Association with Clinical Stage Clin. Cancer Res., June 15, 2009; 15(12): 4085 - 4094. [Abstract] [Full Text] [PDF]

				Y.-C. Lin, L.-Y. Chang, C.-T. Huang, H.-M. Peng, A. Dutta, T.-C. Chen, C.-T. Yeh, and C.-Y. Lin Effector/Memory but Not Naive Regulatory T Cells Are Responsible for the Loss of Concomitant Tumor Immunity J. Immunol., May 15, 2009; 182(10): 6095 - 6104. [Abstract] [Full Text] [PDF]

				A. Wallace, V. Kapoor, J. Sun, P. Mrass, W. Weninger, D. F. Heitjan, C. June, L. R. Kaiser, L. E. Ling, and S. M. Albelda Transforming Growth Factor-{beta} Receptor Blockade Augments the Effectiveness of Adoptive T-Cell Therapy of Established Solid Cancers Clin. Cancer Res., June 15, 2008; 14(12): 3966 - 3974. [Abstract] [Full Text] [PDF]

				N. Monu and A. B. Frey Suppression of Proximal T Cell Receptor Signaling and Lytic Function in CD8+ Tumor-Infiltrating T Cells Cancer Res., December 1, 2007; 67(23): 11447 - 11454. [Abstract] [Full Text] [PDF]

				B. Zhang, T. Sun, L. Xue, X. Han, B. Zhang, N. Lu, Y. Shi, W. Tan, Y. Zhou, D. Zhao, et al. Functional polymorphisms in FAS and FASL contribute to increased apoptosis of tumor infiltration lymphocytes and risk of breast cancer Carcinogenesis, May 1, 2007; 28(5): 1067 - 1073. [Abstract] [Full Text] [PDF]

				M. O. Kilinc, K. S. Aulakh, R. E. Nair, S. A. Jones, P. Alard, M. M. Kosiewicz, and N. K. Egilmez Reversing Tumor Immune Suppression with Intratumoral IL-12: Activation of Tumor-Associated T Effector/Memory Cells, Induction of T Suppressor Apoptosis, and Infiltration of CD8+ T Effectors J. Immunol., November 15, 2006; 177(10): 6962 - 6973. [Abstract] [Full Text] [PDF]

				K. M. Hargadon, C. C. Brinkman, S. L. Sheasley-O'Neill, L. A. Nichols, T. N. J. Bullock, and V. H. Engelhard Incomplete Differentiation of Antigen-Specific CD8 T Cells in Tumor-Draining Lymph Nodes J. Immunol., November 1, 2006; 177(9): 6081 - 6090. [Abstract] [Full Text] [PDF]

				D. T. Shen, J. S. Y. Ma, J. Mather, S. Vukmanovic, and S. Radoja Activation of primary T lymphocytes results in lysosome development and polarized granule exocytosis in CD4+ and CD8+ subsets, whereas expression of lytic molecules confers cytotoxicity to CD8+ T cells J. Leukoc. Biol., October 1, 2006; 80(4): 827 - 837. [Abstract] [Full Text] [PDF]

				M. Koneru, N. Monu, D. Schaer, J. Barletta, and A. B. Frey Defective Adhesion in Tumor Infiltrating CD8+ T Cells J. Immunol., May 15, 2006; 176(10): 6103 - 6111. [Abstract] [Full Text] [PDF]

				A. B. Frey and N. Monu Effector-phase tolerance: another mechanism of how cancer escapes antitumor immune response J. Leukoc. Biol., April 1, 2006; 79(4): 652 - 662. [Abstract] [Full Text] [PDF]

				T. Sun, Y. Zhou, H. Li, X. Han, Y. Shi, L. Wang, X. Miao, W. Tan, D. Zhao, X. Zhang, et al. FASL -844C polymorphism is associated with increased activation-induced T cell death and risk of cervical cancer J. Exp. Med., October 3, 2005; 202(7): 967 - 974. [Abstract] [Full Text] [PDF]

				V. Bronte, T. Kasic, G. Gri, K. Gallana, G. Borsellino, I. Marigo, L. Battistini, M. Iafrate, T. Prayer-Galetti, F. Pagano, et al. Boosting antitumor responses of T lymphocytes infiltrating human prostate cancers J. Exp. Med., April 18, 2005; 201(8): 1257 - 1268. [Abstract] [Full Text] [PDF]

				S. Kusmartsev and D. I. Gabrilovich STAT1 Signaling Regulates Tumor-Associated Macrophage-Mediated T Cell Deletion J. Immunol., April 15, 2005; 174(8): 4880 - 4891. [Abstract] [Full Text] [PDF]

				M. Koneru, D. Schaer, N. Monu, A. Ayala, and A. B. Frey Defective Proximal TCR Signaling Inhibits CD8+ Tumor-Infiltrating Lymphocyte Lytic Function J. Immunol., February 15, 2005; 174(4): 1830 - 1840. [Abstract] [Full Text] [PDF]

				C.-J. Chang, K.-F. Tai, S. Roffler, and L.-H. Hwang The Immunization Site of Cytokine-Secreting Tumor Cell Vaccines Influences the Trafficking of Tumor-Specific T Lymphocytes and Antitumor Efficacy against Regional Tumors J. Immunol., November 15, 2004; 173(10): 6025 - 6032. [Abstract] [Full Text] [PDF]

				S. Radoja, M. Saio, D. Schaer, M. Koneru, S. Vukmanovic, and A. B. Frey CD8+ Tumor-Infiltrating T Cells Are Deficient in Perforin-Mediated Cytolytic Activity Due to Defective Microtubule-Organizing Center Mobilization and Lytic Granule Exocytosis J. Immunol., November 1, 2001; 167(9): 5042 - 5051. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Radoja, S. Articles by Frey, A. B. Search for Related Content PubMed PubMed Citation Articles by Radoja, S. Articles by Frey, A. B.