T Cell-Mediated Tumor Rejection Displays Diverse Dependence Upon Perforin and IFN-γ Mechanisms That Cannot Be Predicted From In Vitro T Cell Characteristics

Liaomin Peng, John C. Krauss, Gregory E. Plautz, Shigehiko Mukai, Suyu Shu and Peter A. Cohen
J Immunol December 15, 2000, 165 (12) 7116-7124; DOI: https://doi.org/10.4049/jimmunol.165.12.7116

Abstract

Experimental pulmonary metastases have been successfully treated by adoptive transfer of tumor-sensitized T cells from perforin knockout (KO) or Fas/APO-1 ligandKO mice, suggesting a prominent role for secretion of cytokines such as IFN-γ. In the present study we confirmed that rejection of established methylcholanthrene-205 (MCA-205) pulmonary metastases displayed a requirement for T cell IFN-γ expression. However, this requirement could be obviated by transferring larger numbers of tumor-sensitized IFN-γ KO T cells or by immunosensitizing sublethal irradiation (500 rad) of the host before adoptive therapy. Extrapulmonary tumors (MCA-205 s.c. and intracranial) that required adjunct sublethal irradiation for treatment efficacy also displayed no requirement for host or T cell expression of IFN-γ. Nonetheless, rejection of MCA-205 s.c. tumors and i.p. EL-4 tumors, but not MCA-205 pulmonary or intracranial tumors, displayed a significant requirement for T cell perforin expression (i.e., CTL participation). The capacity of T cells to lyse tumor targets and secrete IFN-γ in vitro before adoptive transfer was nonpredictive of the roles of these activities in subsequent tumor rejection. Adoptive therapy studies employing KO mice are therefore indispensable for revealing a diversity of tumor rejection mechanisms that may lack in vitro correlation due to delays in their induction. Seemingly contradictory KO data from different studies are reconciled by the capacity of anti-tumor T cells to rely on alternative mechanisms when treated in larger numbers, the variable participation of CTL at different anatomic locations of tumor, and the apparent capacity of sublethal irradiation to provide a therapeutic alternative to host or T cell IFN-γ production.

Since the demonstration >20 years ago that adoptively transferred T cells can mediate tumor rejection, great interest has focused on the mechanisms by which such rejection is accomplished. Early recognition that T cell responses often included Ag-specific lysis of tumor targets led to a prevalent hypothesis that CTL themselves predominantly mediated tumor rejection through direct killing of tumor cells (1, 2, 3, 4, 5, 6). In recent years, the molecular mechanism of classic CTL lysis was found to consist of TCR-triggered T cell secretion of the lytic protein perforin, with resultant pores in the target cell membrane facilitating entry of cosecreted, apoptosis-inducing granzymes (7, 8, 9, 10). An alternative killing mechanism is also available to T cells, employing their expression of CD95 ligand (Fas/APO-1 ligand) to induce target apoptosis, but this second mechanism does not require TCR-triggered initial generation of target cell pores and depends more on target cell CD95 (Fas/APO-1) expression than on MHC-restricted expression of specific Ag (8, 11, 12).

Although considerable evidence indicates that perforin-mediated mechanisms participate in immunosurveillance against tumor carcinogenesis or fresh experimental inocula of tumor cells (6, 9, 13), the relative roles of NK or T cell perforin expression in such immunosurveillance remain unclear, and it has been difficult to prove that direct perforin/granzyme-mediated CTL killing is critical to rejection of established tumors in either animal models or in cancer patients (6, 14). In early adoptive therapy studies performed by Fernandez-Cruz et al., nonlytic rather than demonstrably lytic fractions of T cells proved to mediate rejection of syngeneic rat tumors (15). Barth et al. subsequently demonstrated in mouse studies that rejection of established syngeneic pulmonary metastases by cultured CD8+ tumor-infiltrating lymphocytes (TIL)6 correlated more closely to their capacity to produce cytokines during in vitro tumor exposure than to their much less frequent capacity to lyse the relevant tumor (16). Furthermore, these authors successfully employed anti-IFN-γ Ab or, rarely, anti-TNF-α Ab to neutralize rejection of pulmonary tumors by both lytic and non-lytic TIL (16). Finally, as shown by Schwartzentruber et al., the therapeutic efficacy of TIL cultured from melanoma patients has correlated equally well to their capacity to secrete specific cytokines as to their capacity to lyse tumor targets (17).

Because T cells that are nonlytic in culture can acquire CTL activity in vivo (18, 19), it is desirable to extend mechanistic studies to events that occur in vivo following adoptive transfer. Recent adoptive therapy studies have therefore employed T cells derived from knockout (KO) mice that are persistently compromised with regard to their ability to implement CTL function or cytokine production. Winter et al. demonstrated that anti-CD3-activated T lymphocytes from the tumor-draining lymph nodes (TDLN) of syngeneic mice bearing D5 melanoma or MCA-310 sarcoma could be employed effectively as adoptive therapy of the relevant tumor whether the T lymphocytes were obtained from normal mice, perforinKO mice, or Fas/APO-1 ligandKO mice (14). Although such data demonstrated, in essence, that CTL activity was not an essential mechanism of rejection in these models (20, 21), both D5 and MCA-310 display very low or absent MHC class I expression and/or low natural susceptibility to CTL lysis (14). In addition, these authors focused particularly upon the adoptive therapy of established pulmonary tumors and administered exogenous rIL-2 as an adjunct treatment during adoptive therapy.

Because the majority of the mouse and human tumors studied do not spontaneously hypoexpress MHC class I molecules (22, 23, 24, 25, 26), we have performed adoptive therapy studies using anti-CD-3/IL-2-activated TDLN T cells from syngeneic normal or KO mice to treat weakly immunogenic, MHC class I-expressing tumors. In contrast to the earlier studies, our adoptive therapy experiments were performed with T cell preparations that have repeatedly been demonstrated in the past to achieve rejection of both pulmonary and extrapulmonary tumors, including intracranial, and that do not require coadministration of exogenous rIL-2 to achieve these therapeutic effects (27, 28, 29, 30). Tumor rejection by our T cell preparations has repeatedly been demonstrated to be Ag restricted (i.e., specific for the sensitizing tumor) and to involve participation of both the CD4+ and CD8+ components (27, 28, 29, 30, 31). Within the CD8+ T cell component it is furthermore possible to demonstrate both a helper-dependent and a helper-independent mechanism of tumor rejection (30, 31). We have compared the mechanisms by which adoptively transferred T cells mediate rejection of not only pulmonary but also extrapulmonary tumors, since the latter are typically much less susceptible to such therapy and may have more complex rejection requirements (3, 27, 28, 29).

Consistent with previous reports, we demonstrate a relative IFN-γ requirement, but the lack of a perforin/CTL requirement, when T cell adoptive therapy is directed against a weakly immunogenic sarcoma at the pulmonary location. In contrast, such an IFN-γ requirement was not observed in tumor models in which immunosensitizing sublethal irradiation was necessarily or electively performed before adoptive therapy, including the pulmonary model, suggesting that this adjunct treatment obviated the role of IFN-γ production in tumor rejection. In addition, we provide evidence supporting a significant requirement for T cell perforin expression in instances when adoptive therapy is directed against a variety of extrapulmonary tumors. Finally, we demonstrate that the requirement for T cell perforin or IFN-γ expression during rejection cannot be predicted by the presence or the absence of demonstrable CTL activity or IFN-γ secretion in vitro before adoptive therapy.

Materials and Methods

Mice

Female C57BL/6N (B6) mice were purchased from the Biologic Testing Branch, Frederick Cancer Research and Developmental Center, National Cancer Institute (Frederick, MD). Syngeneic B6 background perforinKO and IFN-γKO mice (C57BL/6-Pfptm1Sdz and C57BL/6-Ifngtm1Ts strains, respectively) were obtained from The Jackson Laboratory (Bar Harbor, ME).

Tumors

Two familiar MHC class I-expressing tumors syngeneic to B6 mice were employed in these studies, the methylcholanthrene (MCA)-induced MCA-205 sarcoma (28, 29) and the EL-4 lymphoma (23). MCA-205 was originally obtained from Jim Yang, Surgery Branch, National Cancer Institute, and cryopreserved aliquots were used to avoid >10 serial passages in mice. EL-4 was obtained from American Type Culture Collection (Manassas, VA). In additional experiments the in vitro passed H-12 sarcoma line, clonally derived and antigenically indistinguishable from MCA-205 (32), was used for TDLN sensitization and adoptive therapy (33).

In vitro analyses

ELISAs to quantitate IFN-γ production and 51Cr release assays to evaluate tumor-specific lysis were performed as described previously (28, 29).

Preparation of TDLN

Serially passaged s.c. MCA-205 tumors were enzymatically digested to obtain a single-cell suspension (28, 29). EL-4 was maintained in tissue culture and cells were harvested for injections. Viable MCA-205 cells (1.5 × 106) or EL-4 cells (5 × 106) were inoculated intradermally in the flanks of healthy B6, or syngeneic perforinKO or IFN-γKO mice. TDLN were sterilely harvested 9 days following tumor inoculation as described previously (28, 29).

In vitro activation of T cells

TDLN were mechanically dispersed to provide tumor-sensitized T cells, which were cultured for 2 days with immobilized anti-CD3, then numerically expanded for an additional 3 days in the presence of rIL-2 as described previously (28, 29).

Adoptive therapy

To establish MCA-205 tumors in healthy syngeneic mice, 3 × 105 viable cells were injected by tail vein into syngeneic normal or, in some cases, syngeneic perforinKO or IFN-γKO mice to establish pulmonary metastases, 1.5 × 106 cells were injected s.c., or 1 × 105 cells were injected intracranially in the right hemisphere of anesthetized mice (27, 28). Alternatively, 5 × 105 viable EL-4 cells were injected i.p. to establish malignant ascites. Three days after tumor inoculation, mice received anti-CD3/IL-2-activated, MCA-205- or EL-4-sensitized TDLN T cells as adoptive therapy (10 or 20 × 106 T cells to treat MCA-205 pulmonary tumors, 50 × 106 T cells to treat MCA-205 s.c. tumors, or 20 × 106 T cells to treat MCA-205/H-12 intracranial tumors or EL-4 i.p. tumors). Mice with s.c. or intracranial tumors always received, and mice with pulmonary tumors electively received, sublethal immunosensitizing irradiation (500 rad) before adoptive therapy (27). None of the treated groups received parenteral rIL-2. All adoptive therapy experiments were performed without coadministration of exogenous rIL-2. Depending on the availability of normal and KO mice, four or five mice were treated under each treatment condition.

Treatment evaluation

Mice with established pulmonary metastases were sacrificed 18 days after tumor challenge, their lungs were insufflated with India ink, and pulmonary metastases were enumerated (28). Mice with established s.c. tumors were evaluated by serial caliper measurements for 40 days or were euthanized sooner when the product of two perpendicular dimensions was >300 mm2 (20). Mice with malignant ascites or intracranial tumor challenges were monitored for disease-free survival and the development of premorbid tumor-related symptoms (prodigious ascites, inanition, or neurologic abnormalities) necessitating euthanasia.

Statistics

Differences in numbers of pulmonary metastases, s.c. tumor dimensions, or disease-free survival among treatment groups were analyzed by the Wilcoxon rank-sum test. A (one-sided) p1 value of <0.025 was considered significant.

Results

In vitro and in vivo properties of EL-4- and MCA-205-sensitized TDLN T cells

As also reported previously, anti-CD3-activated T cells derived from the TDLN of MCA-205-bearing, syngeneic B6 mice displayed specific IFN-γ release when re-exposed to relevant MCA-205 cells in vitro, but did not lyse MCA-205 cells (Fig. 1⇓) despite the sensitivity of MCA-205 cells to lymphokine-activated killer cell-mediated lysis (28, 29). Despite their absence of demonstrable in vitro CTL activity, adoptive transfer of such culture-activated, MCA-205-sensitized TDLN T cells could consistently cure established pulmonary, intracranial, or s.c. MCA-205 tumors in syngeneic mice, but could not cure antigenically irrelevant tumors such as MCA-207 (27, 28, 29) (see below).

FIGURE 1.
FIGURE 1.

Cytokine production and tumor target lysis by anti-CD3/IL-2-activated, MCA-205- or EL-4 sensitized TDLN. For cytokine assays 2 × 106 culture-activated T cells were cocultured 5 × 105 irradiated (5000 rad) stimulator cells (fresh whole-cell enzymatic digest of in vivo passaged MCA-205 or in vitro passaged EL-4) for 24 h as indicated in the figures. Supernatants were harvested, and the concentrations of IFN-γ were measured by ELISA using paired mAb and standards purchased from PharMingen as described previously (28 ,29 ). For the in vitro cytotoxicity assay, tumor cells (1 × 107) were labeled with 51Cr (Na51CrO4, 100 mCi; DuPont, Wilmington, DE) at 37°C for 1 h and washed three times in complete medium. Target cells (1 × 104) were incubated with various numbers of effector cells at 37°C in a volume of 0.2 ml of complete medium for 4 h. The supernatant was collected (Titer-Tek Collecting System, Flow Laboratories, McLean, VA), and the samples were gamma-counted. A, T cells culture-activated from MCA-205-sensitized TDLN; B, T cells culture-activated from EL-4-sensitized TDLN. Line graphs show the percent target lysis calculated as: % lysis = (experimental cpm − spontaneous cpm/maximal cpm − spontaneous cpm) × 100. •, EL-4 targets; ▪, MCA-205 targets. Insets, IFN-γ production by respective culture-activated TDLN T cell group, either without stimulator cells (T cell only) or with EL-4 stimulators (EL-4) or MCA-205 stimulators (MCA-205). Cytokine production is expressed as nanograms per 106 T cells per 24 h. The data shown are representative of five experiments for MCA-205 and three experiments for EL-4.

In contrast, T cells culture-activated from EL-4-bearing, syngeneic normal B6 mice displayed both specific EL-4 lysis and specific IFN-γ release upon re-exposure to EL-4 cells (Fig. 1⇑), and such T cells were highly effective therapy when adoptively transferred to syngeneic mice bearing established i.p. EL-4 tumors (see below).

TDLN T cells from perforinKO mice fail to lyse relevant tumor targets, and TDLN T cells from IFN-γKO mice fail to secrete IFN-γ in vitro

MCA-205- or EL-4-sensitized, culture-activated T cells from normal B6 or perforinKO TDLN, but not from IFN-γKO TDLN, displayed substantial IFN-γ production when re-exposed to relevant tumor cells in vitro (Figs. 2⇓, A and B). T cells culture-activated from normal or IFN-γKO TDLN, but not from perforinKO TDLN, displayed the capacity for specific lysis of EL-4 targets (Fig. 2⇓, C and D). As noted above, in vitro lysis of MCA-205 cells was not observed for culture-activated, MCA-205-sensitized TDLN T cells.

FIGURE 2.
FIGURE 2.

Cytokine production and lytic function of TDLN T cells from normal and KO mice. T cell preparation and activation, cytokine assays, and target lysis assays are described in Materials and Methods and Fig. 1⇑. A and B, IFN-γ production by culture-activated, MCA-205-sensitized (A) or EL-4 sensitized (B) TDLN T cells derived from normal B6 mice (B6 TDLN), syngeneic IFN-γKO mice (IFN-γ KO TDLN), or syngeneic perforinKO mice (perforin KO TDLN). The bars show cytokine production of T cells (nanograms per 106 T cells per 24 h) exposed to the relevant irradiated stimulator cells (MCA-205 in A, EL-4 in B). For both A and B, IFN-γ production by T cells or tumor alone control groups was below assay detectability (<25 pg/ml). T cells from IFN-γKO mice produced no detectable IFN-γ. C and D, Lysis of 51Cr-labeled EL-4 targets by culture-activated, EL-4-sensitized TDLN T cells obtained from normal or KO mice (same groups as in A). T cells from perforinKO mice displayed no lytic activity at any tested E:T cell ratio. The data shown are representative of three experiments for MCA-205 and two experiments for EL-4.

Adoptive therapy of established pulmonary MCA-205 tumors (unirradiated recipients)

When naive mice were challenged i.v. with viable MCA-205 cells to establish pulmonary tumors, subsequent tumor growth was equivalent in either normal B6 or IFN-γKO mice (Fig. 3⇓). When, 3 days following such tumor inoculation, mice additionally received culture-activated T cells, 10 × 106 MCA-205-sensitized T cells derived from normal or perforinKO TDLN proved to be highly effective therapy, whereas 10 × 106 T cells derived from IFN-γKO TDLN were completely ineffective (Fig. 3⇓).

FIGURE 3.
FIGURE 3.

Adoptive therapy of established MCA-205 pulmonary tumors with anti-CD3/IL-2-activated, MCA-205-sensitized TDLN (tumor hosts unirradiated). TDLN were sensitized in normal B6 mice, syngeneic perforinKO mice, or IFN-γKO mice; harvested; and anti-CD3/IL-2 activated as described in Materials and Methods. TDLN T cells (10 × 106) were administered by tail vein 3 days after establishing MCA-205 pulmonary tumors in normal B6 (B6 recipients) or IFN-γKO mice (IFN-γ KO recipients). Mice received no T cells, activated TDLN T cells derived from MCA-205-bearing B6 mice (B6 donor T cells), or activated TDLN T cells derived from MCA-205-bearing perforinKO or IFN-γKO mice (perforin KO donor T cells or IFN-γ KO donor T cells). Fifteen days later mice were sacrificed, and pulmonary tumors were enumerated. The ordinate displays the number of pulmonary tumors. Each filled circle represents a single mouse; there were four or five mice per treatment group. The data shown are representative of two independent experiments. A: A vs B, p1 < 0.0001; B vs C, p1 < 0.0001; B vs D, p1 < 0.0001. B: A vs B, p1 < 0.0001; B vs C, p1 = 1.0.

These results were consistent with prior published reports that adoptively transferred T cells can mediate rejection of established pulmonary tumors in the absence of perforin- or FAS ligand-expressing CTL, and that such tumor rejection can be highly dependent upon IFN-γ production by either adoptively transferred T cells or recipient host cells (14, 16).

The IFN-γ requirement for pulmonary tumor rejection is dose-limited and obviated by sublethal irradiation

In additional experiments, we observed that T cells derived from IFN-γKO TDLN were nonetheless capable of therapeutic efficacy against established MCA-205 pulmonary metastases when they were adoptively transferred in slightly larger numbers (20 × 106) into nonirradiated IFN-γKO hosts (Fig. 4⇓A). When tumor-bearing hosts also received immunosensitizing total body sublethal irradiation (500 rad) before adoptive transfer, the effect of T cells derived from IFN-γKO TDLN was fully equivalent to that of T cells derived from normal B6 TDLN (Fig. 4⇓B).

FIGURE 4.
FIGURE 4.

Adoptive therapy of MCA-205 pulmonary tumors is only relatively IFN-γ dependent. TDLN were sensitized in either normal B6 mice or IFN-γKO mice, harvested, and anti-CD3/IL-2 activated as described in Materials and Methods. TDLN T cells (20 × 106) were administered by tail vein 3 days after establishing MCA-205 pulmonary tumors in IFN-γKO mice (IFN-γ KO recipients). On the day of adoptive therapy, mice were either unirradiated or received total body sublethal irradiation (500 rad), then received no T cells, activated TDLN T cells derived from MCA-205-bearing B6 mice (B6 donor T cells), or activated TDLN T cells derived from MCA-205-bearing IFN-γKO mice (IFN-γ KO donor T cells). Fifteen days later mice were sacrificed, and pulmonary tumors were enumerated. The ordinate shows the number of pulmonary tumors. Each filled circle represents a single mouse; there were four or five mice per treatment group. The data shown are representative of two independent experiments. A: A vs B, p1 < 0.0001; B vs C, p1 = 0.0159; A vs C, p1 = 0.0159. B: A vs B, p1 < 0.0001; B vs C, p1 = 1.0; A vs C, p1 = 0.001.

Adoptive therapy of established s.c. MCA-205 tumors (irradiated recipients)

Compared with pulmonary tumors, treatment of s.c. MCA-205 tumors is relatively refractory to adoptive therapy, requiring higher doses of culture-activated TDLN T cells to achieve cure (27). In addition, adoptive therapy of s.c. tumors is ineffective unless tumor hosts receive total body sublethal irradiation (500 rad) before adoptive therapy (also see Discussion) (27, 34). When naive mice were challenged s.c. with MCA-205 cells, tumor growth was progressive in either normal B6 or IFN-γKO sublethally irradiated mice (Fig. 5⇓). When, 3 days following such tumor inoculation, mice additionally received 500 rad and culture-activated T cells, MCA-205-sensitized T cells derived from normal or IFN-γKO TDLN provided curative therapy. In contrast, T cells derived from perforinKO TDLN could not prevent progressive tumor growth despite a significant growth delay compared with control mice (Fig. 5⇓).

FIGURE 5.
FIGURE 5.

Adoptive therapy of established MCA-205 s.c. tumors with anti-CD3/IL-2-activated, MCA-205-sensitized TDLN. TDLN were sensitized by MCA-205 challenge in either normal B6 or syngeneic KO mice, harvested, and anti-CD3/IL-2 activated as described in Materials and Methods. Three days after establishing MCA-205 s.c. tumors in normal B6 (B6 recipients) or IFN-γKO mice (IFN-γ KO recipients), mice received 500 rad, then adoptive therapy by tail vein consisting of either no T cells or 50 × 106 activated, MCA-205-sensitized TDLN T cells from normal B6 mice (B6 T cells), perforinKO mice (perforin KO T cells), or IFN-γKO mice (IFN-γ KO T cells). Subsequent growth of the s.c. tumors was serially evaluated. The ordinate displays tumor area determined by two perpendicular caliper measurements; the abscissa displays the day following tumor inoculation. Symbols on each line represent the average tumor measurement of each treatment group at particular time points (five mice per treatment group), with the SD displayed. The data shown are representative of two independent experiments. A: A vs B on day 32, p1 = 0.10; B vs C on day 32, p1 = 0.00794. B: A vs B on day 32, p1 = 0.00794; A vs C on day 32, p1 = 0.00794.

These studies demonstrated that tumor rejection can employ perforin/CTL-mediated mechanisms even when such activity is not demonstrable in vitro before adoptive transfer (Fig. 1⇑A). In addition, these studies demonstrated that the capacity of adoptively transferred T cells and/or host cells to produce IFN-γ is not essential to s.c. tumor rejection. It was not possible to ascertain whether the nonessentiality of IFN-γ was a direct consequence of sublethal irradiation, since adoptive therapy of s.c. tumors is ineffective in unirradiated mice (27, 34)

Adoptive therapy of established intracranial MCA-205 tumors (irradiated recipients)

Treatment of intracranial MCA-205 tumors is also relatively refractory to adoptive therapy, requiring higher doses of culture-activated TDLN T cells to achieve cure than in the case of pulmonary tumors (35, 36, 37). In addition, such T cells are typically ineffective for the treatment of intracranial tumors unless the tumor hosts receive local or total body sublethal irradiation (500 rad) before adoptive therapy (also see Discussion) (38). MCA-205-sensitized, culture-activated T cells from either normal or perforinKO TDLN could each provide highly effective adoptive therapy for 3-day intracranial MCA-205 tumors established in either normal B6 or perforinKO syngeneic mice, indicating that perforin gene expression by T cells or host cells may both be nonessential for rejection of intracranial MCA-205 tumors (Fig. 6⇓). In parallel experiments performed with the H-12 clonal derivation of MCA-205, tumor-sensitized, culture-activated T cells from IFN-γKO TDLN provided equally effective adoptive therapy for 3-day intracranial tumors established in either normal B6 or IFN-γKO syngeneic mice, indicating that IFN-γ gene expression by T cells or host cells was also nonessential for rejection of intracranial tumors (32). It was not possible to ascertain whether the nonessentiality of IFN-γ was a direct consequence of sublethal irradiation, since adoptive therapy of intracranial tumors is poorly effective in unirradiated mice (38).

FIGURE 6.
FIGURE 6.

Adoptive therapy of established MCA-205 intracranial tumors with anti-CD3/IL-2-activated, MCA-205-sensitized TDLN. TDLN were sensitized by MCA-205 challenge in either normal B6 or syngeneic KO mice, harvested, and anti-CD3/IL-2 activated as described in Materials and Methods. Three days after establishing MCA-205 intracranial tumors in normal B6 (B6 recipients) or perforinKO mice (Perf KO recipients), mice received 500 rad, then adoptive therapy by tail vein consisting of either no T cells or 20 × 106 activated, MCA-205-sensitized TDLN T cells from normal B6 mice (B6 T cells) or perforinKO mice (perf KO T cells). Mice were followed for disease-free survival; progressive tumor was manifested by focal neurologic deterioration. Symbols on each line show the percentage of mice surviving disease-free (five (A) or four (B) mice per treatment group). The abscissa indicates the day following tumor inoculation. The data shown are representative of two independent experiments. A: A vs B, p1 = 0.00794; A vs C, p1 = 0.00794; B vs C, p1 = 0.690. B: A vs B, p1 = 0.0286; A vs C, p1 = 0.0286.

Adoptive therapy of established i.p. EL-4 tumors (unirradiated recipients)

When naive mice were challenged i.p. with EL-4 cells, subsequent tumor growth was equivalent and lethal in both normal B6 and IFN-γKO mice (Fig. 7⇓). When, 3 days following such tumor inoculation, normal B6 mice additionally received culture-activated T cells, T cells derived from B6 EL-4-sensitized TDLN provided highly effective adoptive therapy in the absence of sublethal irradiation. T cells derived from syngeneic perforinKO TDLN had no detectable therapeutic effect, indicating that T cell perforin expression can be significant to the rejection of established i.p tumor. In contrast, EL-4-sensitized T cells derived from IFN-γKO TDLN provided tumor ablative therapy for a majority of, but not all, recipient IFN-γKO mice (Fig. 7⇓).

FIGURE 7.
FIGURE 7.

Adoptive therapy of established EL-4 i.p. tumors with anti-CD3/IL-2-activated, EL-4-sensitized TDLN (tumor hosts unirradiated). TDLN were sensitized in normal B6 mice, syngeneic perforinKO mice, or IFN-γKO mice; harvested; and anti-CD3/IL-2 activated as described in Materials and Methods. TDLN T cells (20 × 106) were administered by tail vein 3 days after establishing EL-4 i.p. tumors in normal B6 (B6 recipients) or IFN-γKO mice (IFN-γ KO recipients). Mice received no T cells, activated TDLN T cells derived from EL-4-bearing B6 mice (B6 T cells), or activated TDLN T cells derived from EL-4-bearing perforinKO or IFN-γKO mice (perforin KO donor T cells or IFN-γ KO donor T cells). Mice were followed for disease-free survival; progressive tumor was manifested by listlessness, ruffled fur, expanding abdominal girth, and death. Symbols on each line show the percentage of mice surviving disease-free (five mice per treatment group). The abscissa indicates the day following tumor inoculation. The data shown are representative of two independent experiments. A: A vs C, p1 = 0.710; B vs D, p1 = 0.310; A vs B, p1 = 0.00794; C vs D, p1 = 0.00794. B: A vs B, p1 = 0.00794; A vs C, p1 = 0.3670; B vs C, p1 = 0.00794.

Discussion

It is widely accepted that cytokine secretion by T cells can play a prominent or critical role in the rejection of established tumors (reviewed in Ref. 3). When Barth et al. isolated CD8+ TIL from weakly immunogenic MCA-induced sarcomas, the majority of cultured TIL lines lacked the capacity to lyse the relevant tumor target in vitro despite their demonstrated capacity to eradicate established 3-day pulmonary metastases when adoptively transferred in conjunction with exogenous rIL-2 administration (16). However, both lytic and non-lytic TIL released IFN-γ and/or TNF-α when exposed to the relevant tumor in vitro (16). Furthermore, when adoptively transferred to treat established 3-day pulmonary metastases, the therapeutic efficacy of both lytic and non-lytic TIL was ablated by administration of neutralizing Ab to IFN-γ or, in rare cases, neutralizing Ab to TNF-α (16). While these results did not strictly rule out the participation of perforin-mediated CTL lysis in such tumor rejection as an event downstream from cytokine secretion, recent studies in similar models by Winter et al. demonstrated that sensitized T cells from either perforinKO or Fas/APO-1 ligandKO mice were fully capable of rejecting established 3-day pulmonary tumors (14) even in perforinKO or Fas/APO-1 ligandKO hosts, arguing strongly against a role for perforin- or Fas/APO-1 ligand-mediated direct CTL lysis of tumor cells in such models.

The identified mechanisms by which cytokine-producing effector T cells can accomplish tumor rejection in the absence of CTL-mediated tumor lysis are numerous. Individual tumors can be induced to undergo regression simply by exposure to cytokines produced by T cells within their vicinity, due variously to pro-apoptotic and/or anti-angiogenic effects of cytokines such as IFN-γ and TNF-α (reviewed in Ref. 3). Such cytokines also have the capacity to recruit and locally activate accessory cells such as tumoricidal macrophages and lymphokine-activated killer cells which can contact, discriminate, and kill most studied tumor cells regardless of their Ag or MHC expression (reviewed in Ref. 3). Since T cells can be triggered to produce such cytokines when tumor Ag are cross-presented by intratumoral host APC rather than by tumor cells themselves (32, 39, 40, 41, 42), cytokine-mediated tumor rejection has the theoretical potential to proceed even in the complete absence of direct contact between T cells and tumor cells.

The present report supports the evidence that successful T cell-mediated tumor rejection can proceed in the absence of CTL-mediated lysis in the case of experimental pulmonary metastases. Our data concur with the report by Winter et al. that rejection of established 3-day pulmonary metastases proceeds in the absence of T cell perforin expression (14), instead manifesting a relative dependence upon the T1-type cytokine IFN-γ as also described by Barth et al. (16). Like Winter et al., we have also observed that 3-day pulmonary metastases can be effectively treated by adoptive transfer of culture-activated TDLN T cells from Fas/Apo-1 ligandKO mice (data not shown). However, while such data support the nonessentiality of CTL in the rejection of experimental pulmonary metastases, our additional studies disprove the tenet that CTL are never essential for tumor rejection, and furthermore provide no indication that secretion of IFN-γ is ever absolutely essential for tumor rejection.

Even though prior animal studies have indicated a role for endogenous IFN-γ production in the prevention of spontaneous tumor outgrowth (reviewed in Ref. 43), our studies indicate that in the case of extrapulmonary tumors, acute tumor rejection could proceed even when IFN-γ expression is impossible for both adoptively transferred T cells and recipient host cells (Figs. 5⇑ and 7⇑) (32). Even in the case of pulmonary tumors, a seeming requirement for T cell or host cell IFN-γ expression could be bypassed by administering higher numbers of IFN-γKO T cells and immunosensitizing sublethal irradiation. It is therefore evident that effective mechanistic alternatives to IFN-γ production were available in all of our tested tumor models. Furthermore, in certain adoptive therapy experiments (Figs. 5⇑ and 7⇑) a requirement for T cell perforin expression (i.e., CTL activity) was evident despite the nonessentiality of IFN-γ expression. Finally, in the case of MCA-205 intracranial tumors, T cell-mediated rejection could proceed when adoptively transferred T cells and host cells failed to express either IFN-γ or perforin. Such a result may indicate that the expression of either molecule alone was sufficient for intracranial tumor rejection or, alternatively, that both were unnecessary, with the secretion of other cytokines, Fas/APO-1 ligand-mediated lysis, or presently uncharacterized mechanisms proving adequate to secure tumor rejection.

Our comparative analyses therefore indicate that T cell expressions of IFN-γ and perforin each play significant, but not absolute, roles in tumor rejection, which can vary even for the same tumor when it is established at different anatomical sites. It is likely that the relative roles of cytokine secretion and CTL-mediated lysis in tumor rejection are affected by many variables, including the MHC expression and lytic susceptibility of individual tumor lines (26, 44); the ease of inducibility and relative efficiency of other T cell-mediated rejection mechanisms (3); and the dose and phenotype of T cells provided as adoptive therapy (28). Additional relatively minor alterations in experimental conditions can also modulate the observed mechanism of tumor rejection. For example, in contrast to established MCA-205 (wild-type) intracranial tumors, intracranial tumors established from the H-12 clone of MCA-205 display relative resistance to adoptive therapy with perforinKO T cells, which can nonetheless be surmounted by administering larger numbers of perforinKO T cells (data not shown).

The composite data from all of our KO studies are consistent with the possibility that immunosensitizing sublethal irradiation obviates any requirement for host or T cell IFN-γ production in tumor rejection. Evidence for dependence upon T cell IFN-γ expression was apparent in both of our adoptive therapy tumor models where therapeutic effects could be achieved in the absence of sublethal irradiation (Figs. 3⇑ and 7⇑), but not in our models where sublethal irradiation was electively or necessarily administered before adoptive therapy ( Figs. 4–6⇑⇑⇑) (32). The mechanism(s) by which sublethal irradiation facilitates adoptive therapy has been best studied in the case of murine s.c. tumors, where it remains an essential adjunct to T cell adoptive transfer. For successful adoptive therapy of s.c. tumors, sublethal irradiation must be administered to the entire host rather than locally to the tumor bed and can even be applied before tumor inoculation, indicating that normal host cells rather than tumor cells are the essential target of irradiation (34). Host T cells do not appear to be the target of irradiation, since rapid bulk replacement of unirradiated T cells from either normal or tumor-bearing syngeneic mice immediately after irradiation does not interfere with adoptive therapy (27). In addition, sublethal irradiation does not detectably impact the short term trafficking of adoptively transferred T cells into the tumor bed (27). Instead, sublethal irradiation appears to enhance the Ag-presenting function of host cells such as macrophages and dendritic cells within the tumor environment by an as-yet undetermined mechanism (27). Since it is apparent that the immunopotentiating effect of sublethal irradiation is fully operative in the absence of T cell or host cell IFN-γ production (Figs. 4⇑ and 5⇑), it is possible that sublethal irradiation and intratumoral secretion of IFN-γ represent alternative mechanisms for enhancing intratumoral host APC function (29, 45, 46, 47).

Whereas CTL that fail to secrete cytokines such as IFN-γ are virtually never observed in culture, it is quite common to observe cytokine-secreting T cells that are nonlytic in vitro (16, 28, 29). Such findings have fostered wide speculation that T1-type cytokine production may be more essential to T cell-mediated tumor rejection than perforin/granzyme-mediated CTL activity (14). Nonetheless, as demonstrated in the case of MCA-205 rejection, it appears that CTL activity can develop as an essential event only after adoptive transfer, outside the window of observability in culture. Such results reinforce the inadvisability of concluding that CTL mechanisms are inoperative simply because they cannot be demonstrated in vitro. Furthermore, even when CTL activity cannot be demonstrated in vitro, tumor models exist in which T cell and/or host cell expression of IFN-γ is clearly less critical than T cell expression of perforin, including T cell-mediated rejection of MCA-205 s.c. tumors and EL-4 i.p. tumors (Figs. 5⇑ and 7⇑). We are in the process of determining whether, in the case of MCA-205 challenges, induction of T cell perforin expression occurs in the microenvironment of established extrapulmonary tumors but not pulmonary tumors, consistent with the hypothesis that CTL induction may be causally linked to the local failure of other rejection mechanisms.

Footnotes

  • ↵1 This work was supported in part by grants from the National Cancer Institute (CA78263 and CA67324).

  • ↵2 Address correspondence and reprint requests to Dr. Liaomin Peng, Center for Surgery Research, FF-50, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195; E-mail address: pengl{at}ccf.org

  • ↵3 Current address: Ann Arbor Hematology/Oncology Associates, McAuley Cancer Care Building/C139, 5301 Huron River Drive, Ypsilanti, MI 49187.

  • ↵4 Current address: Department of Pediatric Oncology, Yale University Medical Center, 333 Cedar Street, LMP4087, New Haven, CT 06510.

  • ↵5 Current address: 464-504 Santei-cho, Kamigyo-ku, Kyoto 602-0915, Japan.

  • 6 Abbreviations used in this paper: TIL, tumor-infiltrating lymphocytes; KO, knockout; TDLN, tumor-draining lymph nodes; MCA, methylcholanthrene; LAK, lymphokine-activated killers.

  • Received June 29, 2000.
  • Accepted September 21, 2000.

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