Lack of Effector Cell Function and Altered Tetramer Binding of Tumor-Infiltrating Lymphocytes

Ulrike Blohm, Evelyn Roth, Kathrin Brommer, Tilman Dumrese, Felicia M. Rosenthal and Hanspeter Pircher
J Immunol November 15, 2002, 169 (10) 5522-5530; DOI: https://doi.org/10.4049/jimmunol.169.10.5522

Abstract

Tumor-specific CD8 T cell responses to MCA102 fibrosarcoma cells expressing the cytotoxic T cell epitope gp33 from lymphocytic choriomeningitis virus were studied. MCA102gp33 tumors grew progressively in C57BL/6 mice, despite induction of peripheral gp33-tetramer+ T cells that were capable of mediating antiviral protection, specific cell rejection, and concomitant tumor immunity. MCA102gp33 tumors were infiltrated with a high number (∼20%) of CD11b+CD11c− macrophage-phenotype cells that were able to cross-present the gp33 epitope to T cells. Tumor-infiltrating CD8 T cells exhibited a highly activated phenotype but lacked effector cell function. Strikingly, a significant portion of tumor-infiltrating lymphocytes expressed TCRs specific for gp33 but bound MHC tetramers only after cell purification and a 24-h resting period in vitro. The phenomenon of “tetramer-negative T cells” was not restricted to tumor-infiltrating lymphocytes from MCA102gp33 tumors, but was also observed when Ag-specific T cells derived from an environment with high Ag load were analyzed ex vivo. Thus, using a novel tumor model, allowing us to trace tumor-specific T cells at the single cell level in vivo, we demonstrate that the tumor microenvironment is able to alter the functional activity of T cells infiltrating the tumor mass.

Despite the presence of tumor-associated Ags, T cell immune responses against tumors are often ineffective and do not lead to tumor regression. There are several reasons for this. Most tumors are poorly immunogenic because they lack “strong” foreign determinants and are unable to provide costimulatory signals. Thus, many tumors are simply ignored by the immune system because they do not function as professional APCs (1). Tolerance induction (2), incomplete “arming” (3), and impaired functional activity of tumor-infiltrating T cells (4) have also been demonstrated. Stroma surrounding cancer cells may play a critical role in preventing antitumor immunity (5). Furthermore, various tumor cell escape mechanisms prevent efficient recognition of tumor cells by Ag-specific T cells. These include down-regulation of tumor-associated Ags, MHC class I molecules, and proteins involved in Ag presentation (6, 7). Finally, tumor-derived soluble molecules such as TGF-β have been shown to suppress the induction of T cell responses in vitro (8, 9, 10).

To further analyze these processes, we have established a novel murine tumor model using MCA102 fibrosarcoma cells transfected with a minigene encoding residues 33–41 (gp33 epitope) of the glycoprotein from lymphocytic choriomeningitis virus (LCMV).3 The gp33 epitope represents a strong CTL epitope of LCMV (11) and was selected as a model Ag because it allows tracing of gp33/tumor-specific CD8 T cells in normal mice using MHC class I tetramers complexed with the gp33 peptide (12, 13). The gp33 epitope has been used as a tumor-associated model Ag before and, depending on the tumor cell type and Ag expression levels, T cell priming (14, 15), spontaneous tumor regression (16), ignorance (17, 18, 19), or tolerance induction (20) has been observed. The MCA102gp33 tumor model described here differs strongly from these previously described models because progressive tumor cell growth is seen despite induction of systemic gp33/tumor-specific T cell immunity. Thus, the model exhibits striking similarities to the situation in cancer patients where high numbers of tumor-specific T cells were frequently detected, which nonetheless failed to contain tumor progression (21, 22, 23).

Materials and Methods

Mice

C57BL/6 (B6) mice were obtained from our breeding colony and from Harlan Winkelmann (Borchen, Germany). P14 TCR-transgenic (tg) mice (24) (line 327) specific for amino acids 33–41 (gp33 epitope) of the LCMV glycoprotein in association with H-2Db and H8-tg mice (25) ubiquitously expressing the LCMV gp33 epitope as a transgene have been previously described. Female or male mice were used at 8–16 wk of age. Mice were bred and kept in a conventional animal house facility.

Tumors

MCA102 tumor cells (26) were kindly provided by S. Rosenberg (National Institutes of Health, Bethesda, MD) and MCA102gp33 cells were derived from parental tumor cells by gene transfection using the LCMV gp33 minigene as described previously (15). The resulting transfectants were cloned and screened for gp33 expression by CTL assays. The experiments reported in this study were performed with MCA102gp33 clone 3. Cells were cultured in DMEM high glucose supplemented with 10% FCS, glutamine, streptomycin, and penicillin (all from Life Technologies, Gaithersburg, MD). MCA102gp33 cells were kept in culture under G418 (600 μg/ml; Life Technologies) selection. Mice were injected with the indicated number of MCA102gp33 tumor cells in 100 μl PBS s.c. into the flank and tumor size was calculated as the product of bisecting tumor diameters. Mice bearing a tumor with a diameter >15 mm were killed according to animal care regulations.

Peptides and virus

LCMV gp33 (KAVYNFATM), nucleoprotein (NP)396 (FQPQNGQFI), and adenovirus E1A234–243 (SGPSNTPPEI) peptides were purchased from Neosystem (Strasbourg, France). The original cysteine at the anchor position 41 in the LCMV gp33 peptide was replaced by methionine. The LCMV-WE isolate used in this study was originally obtained from R. Zinkernagel (University Hospital, Zurich, Switzerland). Mice were infected i.v. with 200 pfu and viral titers were determined in a virus plaque assay as described (27).

In vivo analysis of cell rejection

Spleen cells from H8-tg mice were incubated at 5 × 106 cells/ml in ice-cold PBS containing 0.5 mM CFSE (Molecular Probes, Eugene, OR) for 10 min at 37°C. The cells were washed once in PBS containing 1% FCS and 108 cells were injected i.v. into recipient mice. The percentage of CFSE+ donor cells among recipient PBL was determined by flow cytometry.

Immunohistochemistry

Tumor sections (5–7 μm) were cut on a cryostat microtome, air dried, fixed in acetone, and blocked with TBS containing 5% mouse serum. Anti-CD8-biotin, anti-CD11b-biotin, and anti-CD11c-biotin (all from BD PharMingen, San Diego, CA) were used as primary mAb followed by avidin-conjugated alkaline phosphatase (StreptAB Complex/AP; DAKO, Hamburg, Germany) and developed with the alkaline phosphatase substrate kit I (Vector Laboratories, Burlingame, CA). Sections were counterstained with Mayer’s hemalum.

Cell isolation

For isolation of tumor-infiltrating cells, tumors were cut into small pieces and digested in PBS containing 0.1% collagenase (Sigma, Taufleirchen, Germany), 0.01% hyaluronidase (Sigma), and 0.002% DNase I (Sigma) for 2 h at 37°C. Undigested material was allowed to settle and released cells were recovered and washed. CD8 T cells were isolated using anti-CD8-conjugated beads (MACS; Miltenyi Biotec, Bergisch-Gladbach, Germany) and MS+ columns. For isolation of macrophages, anti-CD11b-conjugated beads (MACS; Miltenyi Biotec) were used.

T cell cultures and cytotoxicity assay

To test APC function, spleen cells (2 × 106) from P14 TCR-tg or LCMV-immune B6 mice were stimulated with the indicated numbers of CD11b+ cells (purity >95% CD11b+, <0.1% CD11c+), 2 × 105 irradiated (10,000 rad) MCA102gp33 tumor cells, or 2 × 106 B6 spleen cells loaded with 10−6 M gp33 peptide (1 h at 37°C). The cultures were performed in 24-well plates in 1 ml IMDM supplemented with 10% FCS, penicillin/streptomycin, and 0.001 M 2-ME. To induce a CTL response in vitro, 2 × 106 purified tumor-infiltrating lymphocytes (TILs) or 2 × 106 lymphocytes from PBL, spleen, or draining lymph nodes from tumor-bearing mice were stimulated for 5 days in vitro with 2 × 106 gp33 peptide-loaded B6 spleen cells in 24-well plates in 1 ml tissue culture medium. P14 TCR-tg spleen cells (2 × 106) were stimulated similarly for 3 days. Cytolytic activity was tested in 51Cr release assays using EL-4 target cells loaded with gp33 peptide (10−6 M for 2 h at 37°C) or with the control Db-binding adenovirus peptide aa 234–243. To test for Ag-specific cell proliferation, 2 × 106 purified TILs or 2 × 106 spleen cells from P14 TCR-tg mice were labeled with CFSE and stimulated with 2 × 106 gp33 peptide-loaded B6 spleen cells for 3 days in 24-well plates in 1 ml tissue culture medium.

Flow cytometry

Lymphocytes were resuspended in PBS containing 2% FCS and 0.1% NaN3 at a concentration of 106–107 cells/ml, followed by incubation at 4°C for 20 min with 100 μl of properly diluted mAb. For PBL staining 10 U/ml heparin was added to the staining buffer. The following mAb were used: CD8 (clone 53-6.7), CD11b (clone M1/70), CD11c (clone HL3), CD25 (clone 7D4), CD44 (clone IM7), CD45 (clone 30-F11), CD62L (clone MEL-14), CD69 (clone H1.2F3), CD90.1 (clone OX-7), TCR Vα2 (clone B20.1), TCR-αβ (clone H57-597), I-Ab (clone AF6-120.1). All mAb were purchased from BD PharMingen. H-2Db MHC class I tetramers complexed with streptavidin-PE and containing the LCMV gp33 or NP396 peptides were prepared as described (12, 13). The modified Db and β2 plasmids were a kind gift of Dr. J. Altman (Emory University, Atlanta, GA). Tetramer staining was performed at 4°C for 30 min followed by FITC-conjugated anti-CD8 mAb for a further 20 min. TILs or spleen cells were stained with tetramers either directly after cell purification or after preculture (∼106 cells/ml) in tissue culture medium (IMDM plus 10% FCS) for 24 h at 37°C. Addition of IL-2 to the culture medium did not influence tetramer staining results. For direct ex vivo functional analysis of PBL, RBCs were removed by nonfixating PharM Lyse buffer (BD PharMingen) according to the instructions of the manufacturer. For intracellular IFN-γ staining, purified TILs or PBL (105) were stimulated for 5 h in 96-well flat-bottom microtiter plates with gp33 peptide-loaded bone marrow-derived dendritic cells (28) (105) in the presence of 1 μg/ml brefeldin A (Golgistop; BD PharMingen). Cells were then surface stained with anti-CD8-FITC, washed, permeabilized, and restained with PE-conjugated rat anti-mouse IFN-γ mAb (clone XMG1.2; BD PharMingen). Cells were analyzed on a FACSort flow cytometer (BD Biosciences, Mountain View, CA) using CellQuest software. Before analysis of PBL, RBCs were lysed using FACS-Lysing Solution (BD PharMingen).

Results

Growth of MCA102gp33 tumors

To examine the immunogenicity of the gp33 CTL epitope as a tumor-associated Ag, parental and gp33-transfected MCA102 fibrosarcoma cells (106) were transplanted s.c. into unprimed B6 mice. As shown in Fig. 1⇓A, injection of both types of tumor cells resulted in a similar, progressive tumor growth. To exclude the possibility that MCA102gp33 tumor development was due to the selection of gp33 Ag loss variants, tumor cells were isolated and tested in 51Cr release assays. MCA102gp33 cells isolated from tumors were lysed by gp33-specific CTL as efficiently as MCA102gp33 cells kept in in vitro culture, indicating that the gp33 epitope was still expressed in MCA102gp33 tumors growing in B6 mice (Fig. 1⇓B). In LCMV-immune B6 mice possessing gp33-specific memory T cells, MCA102gp33 but not parental MCA102 tumor cells were rejected (Fig. 1⇓C). LCMV infection of MCA102gp33 tumor-bearing B6 mice 10 days after tumor cell injection led to a significant decrease in tumor size after day 15 (Fig. 1⇓D). However, tumor regression was transient and after day 25 progressive tumor growth was observed in all mice. All tumors isolated after this time point represented gp33 Ag loss variants (data not shown). Thus, the gp33 epitope on MCA102 tumor cells serves as a tumor rejection Ag in gp33-primed but not unprimed B6 mice.

FIGURE 1.
FIGURE 1.

Growth of MCA102gp33 tumors. A, Parental and gp33-transfected MCA102 tumor cells (106) were injected s.c. into B6 mice. Tumor size of individual mice, calculated as the product of bisecting tumor diameters, is displayed. B, MCA102gp33 tumors from B6 mice 3 wk after s.c. tumor cell injection were isolated, cultured for 1 week in vitro in the absence of G418, and tested in 51Cr release assays using gp33-specific CTLs from day-8 LCMV-infected B6 mice. Each symbol represents the degree of specific lysis of target cells from one individual tumor at the indicated E:T ratio (right). Parental and MCA102gp33 tumor cell lines kept in in vitro culture were included as controls (left). C, Rejection of MCA102gp33 tumor cells (106) in gp33-primed B6 LCMV memory mice. D, B6 mice bearing day-10 MCA102gp33 tumors were infected with LCMV (solid lines) or were left untreated (dotted lines).

gp33-specific T cell immunity in MCA102gp33 tumor-bearing B6 mice

MHC class I tetramers containing the gp33 epitope (Db-gp33) were used to examine whether inoculation of B6 mice with MCA102gp33 tumor cells could induce gp33-specific T cells. Strikingly, a high number (0.5–8% of CD8) of gp33-tetramer+ cells was detected in PBL of MCA102gp33 tumor-bearing mice (Fig. 2⇓A). To investigate whether this response represented gp33-specific T cell immunity, three functional assays were used.

FIGURE 2.
FIGURE 2.

gp33-specific T cell immunity in MCA102gp33 tumor-bearing B6 mice. A, gp33-tetramer staining of PBL and percentages of gp33-tetramer+ cells of CD8 T cells from individual MCA102gp33 tumor-bearing mice at the indicated time after tumor cell injection. B, Antiviral protection induced by injection of 106 viable (columns 1–3), 107 irradiated (columns 4 and 5), and 107 freeze/thawed (columns 6 and 7) tumor cells. Mice were challenged with LCMV 2 wk after tumor cell injection. Dots represent viral titers in the spleen 4 days after infection in individual mice and the dotted line corresponds to the detection limit of the virus plaque assay. C, Kinetics of adoptively transferred CFSE+ H8-tg donor cells in PBL of the indicated tumor-bearing mice. D, gp33-specific CTL activity of in vitro stimulated lymphocytes from PBL, spleen, and draining lymph nodes (LN) from B6 mice on day 10 (top) and day 20 (bottom) after tumor cell injection. Lymphocytes were stimulated in vitro with gp33 peptide-loaded B6 spleen cells. After 5 days, CTL activity of the cultures was determined in 5-h 51Cr release assays using EL-4 target cells loaded with gp33 (filled symbols) or control adenovirus E1A peptide (open symbols).

First, mice bearing MCA102gp33 tumors were challenged with LCMV 2 wk after tumor cell injection, and viral titers were determined. High viral titers were found in mice bearing parental MCA102 tumors, whereas mice bearing MCA102gp33 tumors had low titers already on day 4 after infection (Fig. 2⇑B). To demonstrate that the observed antiviral protection was due to gp33-specific T cells, the experiment was repeated using H8-tg mice (25) that ubiquitously express the gp33 epitope as a transgene. These mice have a normal T cell repertoire, but due to central tolerance induction they lack gp33-specific T cells. Fig. 2⇑B, column 3, shows that injection of MCA102gp33 tumor cells did not reduce viral titers in H8-tg mice, indicating that the observed antiviral protection in MCA102gp33 tumor-bearing B6 mice was due to the gp33-specific T cell response. Efficient antiviral protection was also obtained when irradiated MCA102gp33 tumor cells were used for immunization (Fig. 2⇑B, columns 4 and 5), whereas injection of nonviable tumor cells (freeze/thawed) failed to induce a gp33-specific response.

Second, the ability of MCA102gp33 tumor-bearing mice to reject gp33 Ag-expressing spleen cells was examined. For these experiments, spleen cells from H8-tg mice, labeled with the vital dye CFSE, were transferred into tumor-bearing mice 2 wk after tumor cell injection, and the kinetics of the transferred donor cells was followed. As shown in Fig. 2⇑C, shortly after adoptive transfer, CFSE+ H8 spleen cells represented 3–5% of host PBL. Afterward, the number of CFSE+ cells decreased over the course of 3 days in mice bearing MCA102gp33 tumors, whereas they persisted in mice bearing parental tumors.

Third, we compared gp33-specific CTL activity of in vitro stimulated lymphocytes from PBL, spleen, and draining lymph nodes of MCA102gp33 tumor-bearing mice. In mice bearing day 10 tumors, gp33-specific CTL activity was predominantly observed in cultures of PBL (Fig. 2⇑D, upper panel). At a later stage (day 20), strong gp33-specific CTL activity was also detected in cultures of spleen and draining lymph nodes (Fig. 2⇑D, lower panel). gp33-specific CTL activity was not detected in T cell cultures from normal mice or mice bearing parental MCA102 tumors (data not shown). Taken together, these data demonstrate that growth of MCA102gp33 tumors induced a strong gp33-specific CTL response capable of mediating antiviral protection and specific rejection of gp33-expressing spleen cells in vivo.

Resistance of MCA102gp33 tumor-bearing mice to a second tumor challenge

The unrestricted growth of MCA102gp33 tumors in the presence of a potent gp33-specific T cell response was unexpected. A kinetic disparity between tumor growth and induction of functional gp33-specific immunity may have occurred, and the tumor has already grown beyond the critical mass that can still be eliminated by T cells (29, 30). To provide T cells with a kinetic advantage, B6 mice bearing 5-day (group 1) or 10-day (group 2) MCA102gp33 tumors on the left flank were challenged with 106 MCA102gp33 tumor cells in the right flank. As shown in Fig. 3⇓A, both groups of tumor-bearing mice were resistant to a second tumor challenge. Concomitant tumor immunity was gp33 Ag-specific because mice bearing MCA102gp33 tumors were not protected against a second tumor challenge with parental MCA102 tumor cells (Fig. 3⇓B). These results demonstrate that gp33-specific T cells induced by the first tumor cell injection could mediate rejection of newly transplanted tumor cells. Thus, the kinetic balance between tumor growth and induction of gp33-specific immunity plays an important role in this model.

FIGURE 3.
FIGURE 3.

Concomitant tumor immunity. A, B6 mice bearing 5-day (group 1) or 10-day (group 2) MCA102gp33 tumors on the left flank were challenged with 106 MCA102gp33 tumor cells in the right flank. Tumor growth in these groups of mice and in a control group injected with tumor cells only on the right flank are depicted. B, Mice bearing MCA102gp33 tumors on the left flank were challenged with 106 parental MCA102 tumor cells in the right flank.

Cross-presentation of gp33 by CD11b+ cells isolated from the tumor mass

MCA102gp33 tumors that grew for 2–3 wk in B6 mice were infiltrated with CD4 and CD8 T cells (2–5%), a high number (∼20%) of CD11b+, and a few (<1%) CD11c+ cells (Fig. 4⇓A). The CD11b+ cells expressed CD45 and MHC class II molecules, but not the dendritic cell marker CD11c, and thus represented most likely tumor-infiltrating macrophages (Fig. 4⇓B). To test whether these cells could present the gp33 epitope to T cells, CD11b+ cells were isolated from the tumor mass (purity >95% CD11b+, <0.1% CD11c+) and were used as APC for in vitro stimulation of responder spleen cells from P14 TCR-tg mice. Irradiated MCA102gp33 tumor cells were included in the assay to judge the direct priming capacity of the tumor cells themselves. As shown in Fig. 4⇓C, top panel, CD11b+ cells isolated from the tumor mass induced gp33-specific CTLs as efficiently as gp33 peptide-loaded B6 spleen cells, whereas MCA102gp33 tumor cells failed to elicit a CTL response. The same results were obtained when spleen cells from LCMV-immune B6 mice were used as responder cells (Fig. 4⇓C, bottom panel). Thus, CD11b+ cells isolated from the tumor mass were able to cross-present the gp33 epitope to CD8 T cells.

FIGURE 4.
FIGURE 4.

gp33 cross-presentation by tumor-infiltrating CD11b+ cells. A, Frozen sections of MCA102gp33 tumors grown for 2–3 wk in B6 mice were stained with anti-CD8, -CD11b, and -CD11c mAb. B, MCA102gp33 tumor-infiltrating cells were stained with mAb specific for the indicated cell surface molecules and analyzed by flow cytometry. C, CD11b+ cells purified from MCA102gp33 tumors were used as APC for in vitro stimulation of responder spleen cells from P14 TCR-tg or LCMV-immune B6 mice. As indicated, irradiated MCA102gp33 tumor cells and gp33 peptide-loaded B6 spleen cells were also examined for APC function in the same assay. The CTL activity of the cultures was determined after 5 days in 5-h 51Cr release assays using EL-4 target cells loaded with gp33 (filled symbols) or control adenovirus E1A peptide (open symbols).

A significant portion of T cells infiltrating MCA102gp33 tumors express TCRs specific for gp33 but exhibit an altered MHC-tetramer binding behavior

Next, the Ag specificity of TILs from MCA102gp33 tumors was determined 2–3 wk after tumor cell injection. Purified CD8 TILs exhibited a highly activated phenotype with up-regulated CD25, CD44, and CD69 and down-regulated CD62L expression (Fig. 5⇓A). Tetramer staining further revealed that 2–3% of CD8 T cells isolated ex vivo from MCA102gp33 tumors appeared to be specific for gp33 Ag. However, the number of gp33-tetramer+ cells increased strikingly when the staining was performed with purified TILs that had been precultured for 24 h in the absence of “contaminating” cells from the tumor mass. Under these conditions, a significant portion of the isolated TILs could be stained with gp33-tetramers, whereas control staining with NP396-tetramers or staining with gp33-tetramers on TILs from parental MCA102 tumors was negative, with or without preculture (Fig. 5⇓B, columns 2 and3). Kinetic analysis showed that the ability of gp33-tetramers to stain purified TILs from MCA102gp33 tumors gradually increased with the duration of in vitro culture (Fig. 5⇓C). However, the intensity of gp33-tetramer staining of TILs was lower than the intensity of PBL from tumor-bearing mice (Fig. 2⇑A). The reduced ability to stain freshly isolated TILs with tetramers was not due to TCR down-regulation, because the cells expressed TCRs at normal levels (Fig. 5⇓A). Thus, a significant portion of the T cells infiltrating MCA102gp33 tumors were specific for the gp33 epitope. However, TILs bound gp33-tetramers only after 24 h preculture in the absence of gp33-expressing/presenting cells.

FIGURE 5.
FIGURE 5.

A significant portion of TILs from MCA102gp33 tumors express TCRs specific for gp33 but exhibit an altered tetramer binding behavior. A, CD8 TILs from MCA102gp33 tumors were purified and stained with mAb reactive with the cell surface molecules indicated. B, gp33- or NP396-tetramer staining of purified CD8 TILs from MCA102gp33 and parental MCA102 tumors directly after isolation (column 1) and after a 24-h preculture (column 2). For better visualization, tetramer staining of precultured TILs is also displayed in histograms (column 3). C, gp33-tetramer staining of TILs from MCA102gp33 tumors, cultured for the indicated time in vitro.

Altered tetramer binding behavior of gp33-specific T cells isolated ex vivo from a gp33-expressing environment

The altered gp33-tetramer binding of TILs could be due to specific factors present in the MCA102gp33 tumor mass or, alternatively, could represent a more general phenomenon where T cells analyzed ex vivo from an Ag-expressing environment cannot efficiently be stained with tetramers. To address this issue, T cells from P14 TCR-tg mice specific for gp33 were adoptively transferred into H8-tg mice that express the gp33 epitope on all MHC class I+ cells (25). As a control, P14 T cells were also transferred into B6 mice. Immediately after cell transfer, both types of recipient mice were infected with LCMV to induce expansion of the transferred donor cells. P14 T cells (Thy1.1+) were traced in the recipient mice (Thy1.2+) using Thy1.1-specific mAb. In B6 mice, P14 T cells could be stained with both mAb specific for the tg TCR Vα2 chain and with gp33-tetramers (Fig. 6⇓A, left panels). In striking contrast, a large fraction of P14 T cells isolated from H8-tg mice could not be stained with gp33-tetramers, despite positive staining with TCR Vα2-specific mAb (Fig. 6⇓A, right panels). Similar to TILs from MCA102gp33 tumors, the ability of gp33-tetramers to stain P14 T cells from H8-tg mice increased significantly after overnight in vitro culture at 37°C but not at 4°C (Fig. 6⇓B). Tetramer staining was specific because P14 T cells were not stained with NP396-tetramers.

FIGURE 6.
FIGURE 6.

Altered tetramer binding behavior of gp33-specific T cells isolated from gp33-expressing H8-tg mice. B6 or H8-tg mice were injected i.v. with 105 Thy1.1+ P14 TCR-tg cells followed by LCMV infection. Eight days after transfer and infection, recipient mice were analyzed by flow cytometry. A, PBL of the mice indicated were stained with Thy1.1-specific mAb (P14 TCR-tg cells) and counterstained with mAb specific for the tg TCR Vα2 chain (top) or with gp33-tetramers (bottom). B, gp33-tetramer staining (right) of P14 TCR-tg cells from LCMV-infected H8-tg recipient mice after a 24-h preculture at 4°C (top) or 37°C (bottom). Staining with NP396-tetramers (left) was performed as a negative control. Dot plots are gated on CD8+ T cells.

TILs in MCA102gp33 tumors lack effector cell functions

The paradox of progressive tumor growth despite the high percentage of gp33-tetramer+ cells within the CD8 subset led us to examine CTL activity, IFN-γ secretion, and proliferative capacity of purified TILs from MCA102gp33 tumors. As shown in Fig. 7⇓, PBL from MCA102gp33 tumor-bearing mice directly used in 5- or 18-h 51Cr release assays efficiently lysed gp33 peptide-loaded target cells. Ex vivo CTL activity of spleen cells was also detected in 18-h 51Cr release assays (∼30% specific lysis at a CD8 to target ratio of 60:1). However, the activity was considerably lower than that of PBL. CTL activity of TILs was <15% in 5-h assays, even after the purified cells had been precultured for 24 h. Under these conditions, most of the cells could be stained with gp33-tetramers (Fig. 5⇑B). In 18-h assays (Fig. 7⇓, right panels), gp33-specific CTL activity of TILs was detectable; however, this activity was ∼10-fold lower on a CD8 cell to target ratio when compared with PBL. IFN-γ secretion of TILs after gp33 Ag stimulation was determined by intracellular cytokine staining. As shown in Fig. 8⇓A, TILs isolated from MCA102gp33 tumors were unable to produce notable amounts of IFN-γ after gp33 Ag stimulation when assayed ex vivo or after a 24-h preculture. In contrast, most of gp33-tetramer+ cells in PBL of MCA102gp33 tumor-bearing mice produced high levels of IFN-γ after gp33 Ag stimulation (Fig. 8⇓B). Finally, the proliferative capacity of TILs after gp33 Ag stimulation was tested. TILs purified from MCA102gp33 tumors were labeled with the vital dye CFSE and were stimulated for 4 days in vitro using gp33 peptide-loaded spleen cells. Under these conditions P14 T cells proliferated vigorously, as indicated by a decrease in their CFSE content. In contrast, TILs failed to divide at all (Fig. 8⇓C), even when the culture medium was supplemented with 40 U/ml IL-2 (data not shown). Furthermore, gp33 stimulation of TILs for 5 days in vitro failed to induce gp33-specific CTL activity, whereas spleen cells from the same tumor-bearing mice specifically lysed target cells when stimulated under the same conditions (Fig. 8⇓D). In short, these data demonstrate that TILs in MCA102gp33 tumor-bearing mice were functionally impaired.

FIGURE 7.
FIGURE 7.

Cytolytic activity of PBL and TILs from MCA102gp33 tumor-bearing B6 mice. CTL activity was determined in 5-h (left) or 18-h (right) 51Cr release assays using EL-4 target cells loaded with gp33 (filled symbols) or control adenovirus E1A peptide (open symbols). PBL were tested directly ex vivo and TILs were tested either directly or after a 24-h preculture.

FIGURE 8.
FIGURE 8.

TILs from MCA102gp33 tumors are unable to secrete IFN-γ and to proliferate after Ag stimulation. A, IFN-γ secretion of TILs was determined by intracellular staining after gp33 Ag stimulation assayed either directly after isolation or after a 24-h preculture. B, gp33-tetramer (left) and intracellular IFN-γ (right) staining of PBL from MCA102gp33 tumor-bearing mice. C, In vitro proliferation of CFSE-labeled TILs stimulated for 4 days with gp33 peptide-loaded B6 spleen cells. The CFSE profiles were gated on CD8+ T cells and P14 TCR-tg cells were included as positive controls. D, Purified TILs from MCA102gp33 tumor-bearing mice were stimulated in vitro with gp33 peptide-loaded B6 spleen cells with (triangles) or without (circles) exogenously added IL-2. After 5 days, CTL activity of the cultures was determined in 5-h 51Cr release assays using EL-4 target cells loaded with gp33 (filled symbols) or control adenovirus E1A peptide (open symbols). CTL activity of cultures using responder spleen cells from the same MCA102gp33 tumor-bearing mice is included for comparison (right).

Discussion

Tumor-specific CTLs are frequently detected among PBL of patients with progressive malignancies (21, 22, 23). The MCA102gp33 tumor model mimics this situation and provides a striking example of progressive tumor growth despite induction of abundant numbers of tumor-specific CD8 T cells. The functional dichotomy of gp33/tumor-specific T cells in the periphery and inside the tumor mass demonstrated here helps to explain this paradox.

The frequency of gp33-specific T cells in tumor-bearing mice was determined by using MHC class I tetramers containing the gp33 epitope. The analysis revealed a high proportion (0.5–8% of CD8) of gp33-tetramer+ cells in PBL. In naive B6 mice, the precursor frequency of cells specific for the gp33 epitope has been estimated to be in the range of 1:104/105 in CD8 T cells (13). Thus, gp33-specific T cells expanded ∼103-fold within 2–3 wk after tumor cell challenge. Clonal expansion of these cells was accompanied by acquisition of effector cell function because MCA102gp33 tumor-bearing mice were protected against LCMV infection and were able to reject gp33-expressing spleen cells in vivo. In this respect, the MCA102gp33 model exhibits similarities to the allogeneic H-2K216 tumor model in which mice bearing K216-positive tumors were able to reject K216-positive skin transplants (31). However, it differs from most other models with defined tumor-associated Ags showing T cell priming (14, 15), tumor regression (16), T cell ignorance (17, 18, 19), or tolerance induction (2, 20, 32).

In a murine colon carcinoma model, peripheral T cells from late stage tumor-bearing mice expressed Ag receptors that contained low amounts of CD3-γ and completely lacked CD3-ζ (33). The model described here differs because peripheral T cells in MCA102gp33 tumor-bearing mice were functional and immune dysfunction was restricted to T cells inside the tumor mass. Interestingly, in patients with colorectal carcinomas CD3-γ levels were low in TILs but only slightly reduced in PBL (34).

The potent induction of a gp33-specific T cell response in tumor-bearing mice raises the question of the mechanism of gp33 Ag presentation. MCA102gp33 tumors were infiltrated with a high number of CD11b+ macrophage phenotype cells. This could be due to soluble factors produced by the tumor cells, specific cell-cell interactions, and/or tumor cell necrosis. Cell debris and possibly also heat shock protein-associated peptides (35) could be taken up by these cells and subsequently cross-presented to CD8 T cells, either inside the tumor mass or after migration to lymphoid organs. Our experiments further showed that tumor-infiltrating CD11b+ cells, but not MCA102gp33 tumor cells themselves, were able to activate gp33-specific T cells in vitro. However, it is doubtful that the systemic gp33-specific T cell immunity observed in tumor-bearing mice was due to Ag presentation by macrophages inside the tumor mass. CD11c+ cells, most likely dendritic cells, were also detected in MCA102gp33 tumors, albeit at a lower frequency. In addition, metastasizing MCA102gp33 cells were found by PCR in local lymph nodes and spleens of tumor-bearing mice (data not shown). Thus, it is more likely that gp33-specific T cell immunity was induced in lymphoid organs of tumor-bearing mice.

In contrast to unprimed B6 mice, LCMV-immune mice or mice already bearing a tumor on the opposite flank rejected MCA102gp33 tumor cells. How can these findings be explained? It has been demonstrated that tumors grow more effectively when transplanted as solid fragments compared with cell suspensions, even though the latter contain more cells (5, 18). Thus, T cells are likely to be more effective against isolated tumor cells compared with tumor cell elimination from a solid tumor mass. In unprimed B6 mice, gp33-tetramer+ T cells were below the detection limit on day 10 after tumor cell injection (Fig. 2⇑A). This time period would allow the transplanted tumor cells to develop a solid tumor mass that is more difficult to control. In mice with an increased frequency of gp33-specific T cells, this kinetic balance is shifted toward protective antitumor immunity.

LCMV infection of B6 mice bearing day-10 MCA102gp33 tumors induced transient tumor regression, followed by outgrowth of gp33 loss variants (Fig. 1⇑D). Due to the low level of viral replication in these mice (Fig. 2⇑B), the number of gp33-tetramer+ T cells in PBL was 10-fold reduced when compared with LCMV-infected control mice (data not shown). Nevertheless, the tumor regression observed indicates that gp33-specific CTL induced by LCMV infection were able to eliminate gp33-expressing tumor cells, even at a later stage of tumor development. It is therefore possible that additional factors triggered by the viral infection, such as induced CD4 T cells and activated APC, help to improve and sustain gp33-specific T cell activity.

The peculiar gp33-tetramer binding behavior of TILs from tumor-bearing mice deserves discussion. Most of the TILs could only be stained with gp33-tetramers after cell purification and a resting period. The impaired tetramer staining of TILs was not due to Ag-induced TCR down-regulation, because ex vivo isolated cells expressed TCRs at normal levels. In contrast, gp33-tetramer+ T cells in PBL could be immediately stained by conventional methods. These circulating T cells probably had less direct contact with gp33-expressing/presenting cells than T cells from the tumor mass. Our results further revealed that “tetramer-negative T cells” were not restricted to TILs in MCA102gp33 tumors, but were also found among gp33-specific P14 T cells isolated from gp33-expressing H8-tg mice. Thus, our data suggest that the inability to stain Ag-specific T cells with tetramers represents a phenomenon that is observed when T cells derived from an environment with high Ag load are analyzed ex vivo.

Failure to stain Ag-specific CD8 T cells with MHC tetramers is not an unprecedented observation. Tetramer-negative Ag-specific CD8 T cells have been described in newborn mice infected with oncogenic polyoma virus (36) and in chronic hepatitis B virus patients (37). Discrepancies between cytokine release or cytolytic activity and tetramer binding have also been found in melanoma patients (38) and have been reported for cultured T cells (39). Furthermore, triggering T cell clones under certain in vitro stimulation conditions has been demonstrated to lead to a loss of tetramer labeling (40). Moreover, tetramer binding has been shown to be an active cellular process requiring cytoskeletal rearrangements (41), and efficient binding also depends on the integrity of lipid rafts on the surface of T cells (42). Thus, interaction of tetrameric MHC molecules with TCRs appears to be influenced by the activation state of the T cell and follows different rules when compared with binding of Abs to cell surface molecules.

The lack of effector cell function of tumor-specific T cells inside the MCA102gp33 tumor provides an explanation for the tumor outgrowth despite potent systemic gp33-specific T cell immunity. It is possible that soluble factors such as TGF-β (8, 9, 10) produced by the tumor cells themselves or by infiltrating cells inhibited the response of the TILs at the tumor site. However, supernatant of MCA102gp33 tumor cells contained only low levels of TGF-β1 or -β2 (∼20 pg/ml, data not shown). Alternatively, permanent stimulation of gp33-specific T cells by gp33-expressing tumor cells and by gp33 cross-presenting macrophages in the absence of costimulatory signals may lead to the observed loss of effector cell function of the TILs. In this respect, it is noteworthy that neither MCA102gp33 tumor cells nor tumor-infiltrating macrophages express costimulatory molecules such as CD80, CD86, or ICAM-1 (data not shown). The activated phenotype and the lack of effector function of TILs in MCA102gp33 tumors are reminiscent of virus-specific “Sisyphean” CD8 T cells found in persistently infected hosts (43). Similarly, P14 T cells isolated from LCMV-infected H8-tg mice ubiquitously expressing gp33 have been shown to exhibit an activated phenotype and to be anergic (25). As demonstrated in this study, these T cells also display an altered gp33-tetramer staining behavior similar to TILs from MCA102gp33 tumors (Fig. 6⇑). Thus, we favor the view that gp33-specific T cells activated in lymphoid organs of MCA102gp33 tumor-bearing mice “exhaust” inside the tumor mass due to the high Ag load present mainly on nonprofessional APC.

In conclusion, the present study provides a striking in vivo example of how a tumor can grow progressively despite potent systemic tumor-specific T cell immunity. The model helps us to understand failures in tumor regression in patients despite successful induction of tumor-specific CTLs in the periphery. Our study emphasizes the importance of understanding the local mechanisms that prevent destruction of an established tumor by T cells.

Acknowledgments

We thank Drs. S. Batsford and S. Ehl for comments on the manuscript, Dr. S. Rosenberg for MCA102 tumor cells, Dr. M. Weller for CCL-64 cells, and Dr. J. Altman for modified H-2Db and β2 microglobulin constructs. We also thank Oliver Schweier for generation of MHC tetramers, Marlies Rawiel for expert technical assistance, and Theresa Treuer, Rainer Bronner, and Thomas Imhof for animal husbandry.

Footnotes

  • 1 This work was supported by the Deutsche Krebshilfe (10–1724-Pi).

  • ↵2 Address correspondence and reprint requests to Dr. Hanspeter Pircher, Institute of Medical Microbiology and Hygiene, Department of Immunology, Hermann-Herder-Strasse 11, University of Freiburg, D-79104 Freiburg, Germany. E-mail address: pircher{at}UKL.uni-freiburg.de

  • ↵3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; tg, transgenic; TIL, tumor-infiltrating lymphocyte; NP, nucleoprotein.

  • Received June 14, 2002.
  • Accepted September 24, 2002.

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