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Keywords:

  • Cytotoxic T cells;
  • Rodent;
  • Tumor immunity;
  • Viral

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

We determined the efficacy of in vitro expanded P14 TCR transgenic CD8 T cells to mediate tumor cell elimination and to protect against viral infection in mice. Contrary to previous studies, an adoptive transfer model without lymphodepletion, vaccination or cytokine treatment was used. Antigen-activated P14 T cells cultured in IL-2-containing medium for 7 days (P14IL-2) exhibited potent effector cell functions in vitro but did not confer protection against melanoma growth or viral infection. In contrast, P14 T cells cultured in IL-15 (P14IL-15) were highly effective in vivo although they displayed only moderate effector functions in vitro. Therapeutic efficacy correlated with the survival of the transferred T cells in the recipients: P14IL-2 cells disappeared rapidly whereas P14IL-15 cells persisted for prolonged time. Decreasing the IL-2 concentration in the culture media improved in vivo survival and efficacy but also lowered the cell yield of the cultures. Finally, we could extend the findings with monoclonal P14 T cells to polyclonal CD8 T cells. Thus, in vitro expansion of antigen-specific CD8 T cells in IL-15 allowed the generation of substantial numbers of T cells without inducing terminally differentiated effector cells that turned out to be unfavorable in the transfer model examined here.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Adoptive transfer of antigen-specific CD8 T cells into hosts represents a promising approach to treat tumors and viral infections 1–4. To generate sufficient numbers of T cells for this therapy, T cells have to be stimulated and expanded in vitro. In most protocols used today, IL-2 is added to the culture medium to ensure T-cell proliferation, differentiation and survival. Besides IL-2, IL-15 also supports CD8 T-cell growth 5; however, the phenotype and the functional activity of IL-2- versus IL-15-cultured CD8 T cells differ considerably. Manjunath et al. 6 were the first to show that CD8 T cells from P14 TCR-tg mice specific for gp33 epitope of lymphocytic choriomeningitis virus (LCMV) acquired an effector memory phenotype with high cytolytic activity when cultured in the presence of IL-2 whereas addition of IL-15 led to a central memory phenotype with low effector cell functions. Further analysis in the same TCR transgenic system revealed that these two cytokines were equivalent mitogens for antigen-stimulated CD8 T cells but that IL-2 was more potent in inducing amino acid uptake and protein synthesis 7.

For adoptive T-cell therapy, it is important to generate CD8 T cells in vitro, which are efficient in inducing tumor regression or virus clearance in vivo. The degree of specific target cell lysis and the amount of antigen-triggered IFN-γ production are frequently used to predict the efficacy of CD8 T cells in vivo. However, several recent studies in the pmel-1 TCR-tg model specific for the self/tumor antigen gp100 of B16 melanoma cells indicated that antigen-stimulated CD8 T cells with a less differentiated phenotype were superior in anti-tumor activity compared with more differentiated cells 8–10. In these studies, in vitro activated pmel-1 CD8 T cells were transferred into sublethally irradiated hosts followed by gp100 vaccination using recombinant fowl pox virus and in vivo IL-2 treatment. Lymphodepletion is known to enhance the efficacy of adoptively transferred CD8 T cells but renders the host more susceptible to infections. In addition, IL-2 treatment is known to cause severe dose-limiting toxicities in patients 11. It is therefore important to determine whether CD8 T cells with a less differentiated phenotype are also more efficient in anti-tumor or anti-viral activity in adoptive transfer models without lymphodepletion, vaccination or cytokine treatment. To address this question, we compared the efficacy of IL-2- versus IL-15-stimulated P14 T cells (P14IL-2, P14IL-15) that exhibited effector or central memory phenotypes in tumor cell elimination and anti-viral activity after adoptive transfer into non-irradiated recipient mice without further treatments. In addition, polyclonal CD8 T cells from LCMV immune B6 mice stimulated with CD3- and CD28-specific antibodies in vitro were also used to address this issue.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Phenotype and functional activity of P14IL-2 and P14IL-15 cells

P14IL-2 and P14IL-15 cells were generated in vitro using the protocol originally described by Manjunath et al. 6. Briefly, CD8 T cells from P14 TCR-tg mice specific for the LCMV gp33 epitope were stimulated with gp33 peptide for 2 days to induce lymphoblasts. Afterwards, the cells were cultured in medium containing IL-2 (20 ng/mL) or IL-15 (50 ng/mL) for an additional 7 days. P14IL-2 cells generated by this protocol showed a blastoid morphology and an increased granularity in the flow cytometer compared with P14IL-15 cells (Fig. 1A, top). P14IL-2 cells exhibited an effector phenotype with almost complete downregulation of CD62L and CCR7 whereas P14IL-15 cells showed a central memory phenotype with significant expression of CD62L and CCR7 (Fig. 1A, bottom). Analysis of the IL-2 and IL-15 receptor subunits (Fig. 1B) revealed that P14IL-2 cells expressed considerably higher levels of the IL-2 receptor α chain (CD25) than P14IL-15 cells. Expression of the IL-2/15 receptor β chain (CD122) and of the common γ chain (CD132) was comparable in both cell types. The IL-15R α chain that is involved in IL-15 presentation but not in signaling was found at low levels on P14IL-2 cells but was undetectable on P14IL-15 cells perhaps due to ligand-induced downregulation. In contrast to effector cells generated in the pmel-1 TCR-tg system 8, KLRG1 was not expressed by in vitro stimulated P14 T cells (data not shown).

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Figure 1. Phenotype of P14IL-2 and P14IL-15 cells. P14 T cells were stimulated with gp33 peptide for 2 days and then cultured in the presence of either IL-2 (20 ng/mL) or IL-15 (50 ng/mL) for the next 7 days. Afterwards, the cells were analyzed by flow cytometry using the mAb indicated. (A) The dot plots showing expression of CD8 and TCR Vα2 were gated on total live cells using the regions indicated in the FSC/SSC plots. The plots showing expression of CD62L and CCR7 were gated on CD8 T cells. The numbers in the quadrants of the dot plots represent the percentages of the gated population. (B) Expression of the IL-2 and IL-15 receptor subunits determined by the indicated mAb (filled histograms). Open histograms show negative control staining. Histograms were gated on CD8 T cells. Representative results from one out of two to three experiments are shown.

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P14IL-2 cells showed high cytolytic activity against gp33 peptide loaded EL-4 and gp33 expressing B16 target cells in standard 51Cr release assays (Fig. 2A and B). Target cell killing was antigen-specific since target cells not presenting the nominal antigen were not lysed. P14IL-15 cells showed only moderate lytic activity against EL-4/gp33 target cells and almost no lytic activity against B16gp33 melanoma cells. On a cell per cell basis, the cytolytic activity of P14IL-2 cells was ∼30-fold higher than that of P14IL-15 cells. When antigen-triggered IFN-γ production was examined, P14IL-2 were also more potent than P14IL-15 cells; however, the difference in the percentages of IFN-γ secreting cells was less prominent (two- to threefold, Fig. 2C). These data are well in line with previous reports that exposure of antigen-stimulated P14 or pmel-1 T cells to IL-2 or IL-15 generates effector or central memory-like CD8 T cells, respectively 6, 8–10, 12. We also noted that P14IL-2 and P14IL-15 cells differed in functional CD95L/Fas ligand expression since co-culture of P14IL-2 cells and L1210 target cells expressing CD95/Fas resulted in antigen-independent cell killing whereas co-culture of the same target cells and P14IL-15 cells had no effect (Fig. 2D). Cell-mediated lysis under these conditions was CD95/Fas-dependent since parental L1210 target cells were not lysed by P14IL-2 cells.

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Figure 2. Functional activity of P14IL-2 and P14IL-15 cells in vitro. The cytolytic activity of P14IL-2 and P14IL-15 cells was tested in 5 h 51Cr-release assays using EL-4 target cells loaded with gp33 or control peptide (A). In (B), B16 cells expressing the gp33 epitope (B16gp33) and parental B16 cells were used as targets. (C) IFN-γ production determined by intracellular cytokine staining of P14 T cells with or without gp33 peptide restimulation. The dot plots were gated on total live cells and the numbers in the quadrants represent the percentages of the gated population. (D) CD95/Fas-dependent cell-mediated lysis determined in 5 h 51Cr-release assays using CD95/Fas-transfected and parental L1210 target cells in the absence of gp33 peptide. Shown are representative results from two independent experiments.

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Only P14IL-15 cells mediate B16gp33 tumor-cell elimination in vivo

The therapeutic efficacy of P14IL-2 and P14IL-15 cells was assessed in the B16gp33 melanoma lung metastases model. Pulmonary metastases were induced in B6 mice by i.v. injection of B16gp33 melanoma cells. Two days later, P14IL-2 or P14IL-15 cells were transferred i.v. into the tumor-bearing mice, and the numbers of lung metastases were evaluated two weeks after tumor cell transfer. As shown in Fig. 3, adoptive transfer of P14IL-15 cells caused a massive reduction in the number of pulmonary metastases compared with animals that received no treatment. In striking contrast, adoptive transfer of the same number of P14IL-2 cells completely failed to lower the number of pulmonary metastases. Similarly, transfer of naive CD8 T cells from P14 TCR-tg mice had no effect on the number of metastases (data not shown). Thus, these data demonstrate that P14IL-15 cells were highly effective in tumor elimination in vivo although they exhibited only moderate effector functions in vitro whereas P14IL-2 cells with potent effector functions in vitro displayed no therapeutic efficacy in vivo under these experimental conditions.

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Figure 3. Anti-B16gp33 tumor response of P14IL-2 and P14IL-15 cells in vivo. Pulmonary metastases were induced in B6 mice by i.v. injection of 106 B16gp33 melanoma cells. Two days later, 5×106 P14IL-2 or P14IL-15 cells were transferred i.v. into the tumor-bearing mice. Mice were analyzed 2 wk after tumor cell transfer. (A) Representative pictures of lungs from mice without treatment (top) and after adoptive transfer of P14IL-2 or P14IL-15 cells. (B) Number of macroscopically visible foci on the lung surface of control mice (w/o transfer) and of recipients of P14IL-2 or P14IL-15 cells. Two independent experiments are shown. Circles and triangles represent values from individual mice.

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Prolonged survival of P14IL-15 cells in recipients after adoptive transfer

To rationalize the striking difference in anti-tumor activity of P14IL-2 and P14IL-15 cells in vivo, the fate of the transferred P14 T cells in the recipient mice was determined. In the first experiments, P14 T cells (Thy1.1+) were traced in the recipient mice using Thy1.1-specific mAb (Fig. 4A). Twenty-four hours after intravenous T-cell transfer, both P14 T-cell populations were detected in the lung albeit the frequency of P14IL-2 cells was about twofold lower compared with P14IL-15 cells (0.4 versus 0.7% of total lymphocytes). After 48 h, the frequency of P14IL-15 cells remained unchanged whereas P14IL-2 cells were no longer detectable. In the spleen, P14IL-2 cells were below detection limit (<0.1%) at both time points as opposed to P14IL-15 cells that represented ∼1% of total splenocytes.

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Figure 4. Survival of P14IL-2 and P14IL-15 cells after adoptive transfer in vivo. (A) 5×106 P14IL-2 or P14IL-15 cells were injected i.v. into B6 mice and lymphocytes from lung and spleen of the recipient mice were analyzed 24 and 48 h after transfer. P14 T cells were traced by Thy1.1-specific mAb. The dot plots were gated on total lymphocytes and the numbers in the quadrants represent the percentages of the gated population. (B) P14IL-2 and P14IL-15 cells were labeled with CMTMR and CFSE, respectively, and co-transferred in equal numbers (each 5×106) into the same recipient mice via the intravenous route. At different time points after transfer, lymphocytes from the organs indicated were isolated and the proportion of P14IL-2 and P14IL-15 cells of total CD8 T cells was determined. Circles represent values from individual mice. The dot plots shown were gated on CD8 T cells and the numbers in the quadrants represent the percentages of the gated population. (C) P14IL-2 and P14IL-15 cells were labeled with CFSE and CMTMR, respectively, and injected i.p. in equal numbers (each 5×106) into the same recipient mice. At the time points indicated, peritoneal leukocytes were analyzed. The dot plots were gated on CD8 T cells and the numbers in the quadrants represent the percentages of the gated population. Data from one representative experiment out of four are shown.

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To compare the survival of the two donor T-cell populations more directly, P14IL-2 and P14IL-15 cells were labeled with different dyes such as CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) and CFSE, respectively and co-transferred in equal numbers into the same recipient mice. At different time points after T-cell transfer, lymphocytes from lung, liver, blood, spleen and lymph nodes were isolated and the proportion of P14 T cells of total CD8 T cells was determined (Fig. 4B). In the lung, P14IL-2 and P14IL-15 cells were present at comparable frequencies (20–40% of total CD8 T cells) 2 h after transfer. Afterwards, the frequency of P14IL-2 cells dropped rapidly and the cells were no longer detectable (<0.1%) 5 days later. The frequency of P14IL-15 cells in the lung also decreased but the cells were still present (5–7% of total CD8 T cells) 5 days after transfer. In the liver, P14IL-15 cells were present at high frequencies (50–60%) 2 h after transfer and this value dropped to∼5% after 5 days. In contrast, P14IL-2 cells could only be detected within the first 48 h after transfer. At any time point tested, P14IL-2 could not be recovered from spleen, peripheral blood or peripheral lymph nodes whereas P14IL-15 cells were present in sizable numbers (2–8% of total CD8 T cells) in these organs also at later time points.

The failure of P14IL-2 cells to enter the circulation after i.v. injection could be due to mechanical sequestration of the blastoid cells in microvessels of lung and liver. Therefore, we also compared the survival of the two P14 T-cell populations in the peritoneal cavity after i.p. injection (Fig. 4C). Two hours after the injection, comparable numbers of P14IL-2 and P14IL-15 cells, labeled with different dyes, were recovered. Afterwards, the proportion of P14IL-2 cells in peritoneal leukocytes dropped rapidly resulting in a five- to tenfold increase in the ratio of P14IL-15 to P14IL-2 cells 24 and 48 h after transfer. Taken together, these results indicated that the vast majority of P14IL-2 cells died rapidly after adoptive transfer in striking contrast to P14IL-15 cells that persisted in the recipient mice in sizable numbers for prolonged time.

Factors determining survival of activated P14 T cells

Cytokine withdrawal may play a crucial role in the rapid disappearance of P14IL-2 cells in vivo. To mimic this situation in vitro, apoptosis of P14IL-2 and P14IL-15 cells cultured for additional 2 days in the presence or absence of cytokines was determined (Fig. 5A). In the absence of cytokines, the number of apoptotic cells, determined by Annexin V staining, was considerably higher in P14IL-2 compared with P14IL-15 cultures (89 versus 52% apoptotic cells). Addition of IL-2 prevented apoptosis in both cultures (22 and 11% apoptotic cells) whereas IL-15 could only partially prevent apoptosis of P14IL-2 cells compared with a nearly complete rescue of P14IL-15 cells (48 versus 10% apoptotic cells). Next, we determined whether high dose IL-2 treatment of recipient mice could prolong persistence of the transferred P14IL-2 cells in vivo. Recombinant IL-2 was administered twice daily on days 0, 1 and 2 after T-cell transfer using a protocol established by Restifo and co-workers 8. At day 2 after transfer, the frequencies of P14IL-2 cells in the lung were increased about twofold by the IL-2 treatment but at day 5, P14IL-2 cells decreased to similar low levels in both treated and untreated mice (Fig. 5B). In the blood, P14IL-2 cells were below detection limit at both time points. These data indicate that high dose IL-2 treatment in this transfer system using non-irradiated recipients transiently enhanced the numbers of P14IL-2 cells in the lung but failed to improve their survival in the periphery.

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Figure 5. Factors determining survival of activated P14 T cells. (A) Rapid apoptosis of P14IL-2 cells after cytokine withdrawal in vitro. P14IL-2 and P14IL-15 cells, cultured for 7 days in IL-2 and IL-15, respectively, were re-cultured for additional 2 days with or without the indicated cytokines (20 ng/mL IL-2; 50 ng/mL IL-15). The percentages of apoptotic cells were determined by Annexin V staining. The dot plots were gated on total cells and the numbers in the regions represent the percentages of the gated population. Data from one representative experiment out of two are shown. (B) High dose IL-2 treatment in vivo. P14IL-2 cells (5×106) were transferred into B6 mice and recombinant IL-2 was administered twice daily on days 0, 1 and 2 after T-cell transfer. The percentages of P14IL-2 cells of total CD8 T cells in lung and blood of the recipient mice were determined at days 2 and 5 after transfer. Circles represent values from individual mice. (C) Short-term culturing of P14IL-2 cells improves cell survival after transfer. Activated P14 T cells were cultured in the presence of IL-2 (20 ng/mL) for 3, 5 and 7 days before adoptive transfer. The percentages of the transferred P14IL-2 cells (5×106) of total CD8 T cells were determined in the blood of the recipient mice at the indicated time points after transfer. Pooled data from two experiments are shown and circles represent values from individual mice.

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A previous study by Malek co-workers using OT-1 T cells indicated that the period of time of in vitro expansion in IL-2 played a crucial role in the survival after adoptive transfer 13. To examine this issue in our system, activated P14 T cells were cultured in vitro in the presence of IL-2 (20 ng/mL) for 3, 5 and 7 days before adoptive transfer. The numbers of the recovered P14IL-2 cells in the recipient mice inversely correlated with the period of time of in vitro culturing in IL-2 (Fig. 5C). Activated P14 T cells expanded for 3–5 days in vitro with 20 ng/mL of IL-2 were readily detectable in the recipient mice whereas activated P14 T cells cultured for 7 days failed to survive after transfer.

P14 T cells cultured in low concentrations of IL-2 resemble P14IL-15 cells

Next, we determined whether P14 T cells exhibiting a central memory phenotype could be generated by decreasing the amount of IL-2 in the culture medium. Indeed, this proved to be the case. P14 T cells cultured in low amounts of IL-2 (0.2–1 ng/mL) exhibited a phenotype comparable to IL-15-stimulated cells with increased CD62L and decreased CD25 expression (Fig. 6A, top and middle). It is, however, important to emphasize that absolute numbers of P14 T cells recovered from these cultures were five- to tenfold lower compared with cultures with saturating amounts of IL-2 (5–20 ng/mL) or IL-15 (50 ng/mL) (Fig. 6A, bottom).

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Figure 6. P14 T cells cultured in low concentrations of IL-2 resemble P14IL-15 cells. (A) P14 T cells were stimulated with gp33 peptide for 2 days and then cultured in IL-2 or IL-15 as indicated for 7 days. CD62L and CD25 cell surface expression and absolute numbers of recovered P14 T cells are depicted. Input number of P14 T cells: 1×106. Data from one representative experiment out of three are shown. (B) P14 T cells (107), cultured in IL-2 or IL-15 as indicated, were transferred into B6 mice. Symbols represent the percentages of donor P14 T cells of total CD8 T cells in the blood of the recipient mice. Data are from one out of two experiments. (C) P14 T cells (106), cultured in IL-2 or IL-15 as indicated, were transferred into B6 mice carrying pulmonary metastases of B16gp33 melanoma cells. The number of macroscopically visible foci in the lung was determined 2 wk after tumor cell transfer. Symbols represent values from individual mice. (D) P14 T cells (107), cultured in IL-2 or IL-15 as indicated, were transferred into B6 mice that were challenged with LCMV 9 days later and viral titers were determined. Symbols represent values from individual mice. Data from one representative experiment out of two are shown.

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To determine whether P14 T cells cultured under low IL-2 (1 ng/mL) conditions show a better in vivo survival than cells cultured in 20 ng/mL of IL-2, adoptive transfer experiments were performed. Strikingly, only P14 T cells kept under low IL-2 conditions or cells cultured in IL-15 (20 or 50 ng/mL) were detectable in the recipient mice after transfer (Fig. 6B). The therapeutic efficacy of P14IL-2 cells cultured in low amounts of IL-2 was further assessed in the B16gp33 melanoma lung metastases model. The data in Fig. 6C show that only P14IL-15 or P14IL-2 cells cultured in low concentrations of IL-2 (1 ng/mL) were capable of lowering the number of pulmonary metastases.

Finally, we tested the anti-viral efficacy of the different P14 T-cell populations using the LCMV infection model. Recipient mice were challenged with LCMV 9 days after transfer and viral titers in the spleen were determined 4 days later (Fig. 6D). The results revealed that transferred P14 T cells cultured in IL-15 (20 or 50 ng/mL) or in low dose IL-2 (1 ng/mL) conferred almost complete anti-viral protection whereas P14 T cells cultured in 20 ng/mL of IL-2 did not reduce viral titers. Thus, cultivating activated P14 T cells in IL-15 allowed the generation of substantial numbers of T cells in vitro that exhibited high anti-tumor and anti-viral activity. When saturating amounts of IL-2 were used, effector cells with poor in vivo survival and lack of efficacy were generated. A decrease in the IL-2 concentrations induced central memory phenotype cells with enhanced survival and improved anti-tumor and anti-viral efficacy but it also resulted in a significantly lower cell yield of the T-cell cultures.

Anti-viral protection by IL-15-stimulated polyclonal CD8 T cells

To determine whether the findings in the P14 transfer model could be extended to a polyclonal system, spleen cells from LCMV immune B6.Thy1.1+ mice were stimulated for 2 days with magnetic beads coated with anti-CD3/CD28 antibodies. After removal of the beads, the cells were cultured in a medium containing IL-2 (20 ng/mL) or IL-15 (50 ng/mL) for additional 7 days. Similar to the P14 system, anti-CD3/CD28 stimulated T cells cultured in IL-2 (B6IL-2) showed an increased granularity and enhanced CD62L downregulation in comparison with B6IL-15 cells (Fig. 7A, top). The frequencies of LCMV-specific CD8 T cells in these cultures, determined by MHC class-I tetramers (Db-gp33 and Db-np396) staining, were comparable (Fig. 7A, bottom). At the end of the 9-day culture period, equal numbers (5×106) of B6IL-2 and B6IL-15 cells containing predominantly (∼80%) CD8 T cells were adoptively transferred (i.v.) into non-irradiated mice and traced with Thy1.1-specific antibodies. Similar to the P14 model, only B6IL-15 cells were detected in the peripheral blood of the recipient mice 6 days after transfer (Fig. 7B).

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Figure 7. Characteristics of anti-CD3/CD28-activated polyclonal CD8 T cells cultured in the presence of IL-2 or IL-15. (A) Spleen cells from LCMV immune B6.Thy1.1 mice were stimulated for 2 days with anti-CD3/CD28 mAb coated on magnetic beads and then cultured in the presence of either IL-2 (20 ng/mL) or IL-15 (50 ng/mL) for the next 7 days. These T-cell cultures, termed B6IL-2 and B6IL-15, were stained with CD62L- and CD8-specific mAb and the frequencies of LCMV gp33- and LCMV np396-specific T cells were determined by MHC-class I-tetramers. Shown are representative dot plots gated on total live cells and the numbers in the quadrants represent the percentages of the gated population. The bars show mean values of % tetramer+ cells of CD8 T cells derived from four independent experiments. (B) B6IL-2 and B6IL-15 cells (5×106) were injected i.v. into recipient mice and 6 days after transfer, the proportion of donor Thy1.1+ CD8 T cells was determined in the blood. Shown are representative dot plots gated on peripheral blood lymphocytes and the numbers in the quadrants represent the percentages of the gated population. The bars show mean values from 15 recipient mice derived from four independent experiments.

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To determine whether the transferred T cells can mediate anti-viral protection, the recipient mice were challenged with LCMV and viral titers were determined. Furthermore, clonal expansion of the transferred donor T cells after LCMV infection was analyzed in another group of mice. The experiments revealed that recipients of B6IL-15 cells had∼103-fold lower viral titers compared with control mice without transfer. Recipients of B6IL-2 cells exhibited a high viral load comparable to control mice (Fig. 8A). In addition, we observed a more vigorous expansion of LCMV-specific B6IL-15 cells in the recipient mice after infection compared with B6IL-2 cells. Together, these data demonstrate that polyclonal T cells from LCMV-immune mice activated by anti-CD3/CD28 antibodies and cultured in the presence of IL-15 exerted potent anti-viral protection in contrast to T cells expanded in the presence of IL-2 (Fig. 8B).

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Figure 8. Antiviral activity of anti-CD3/CD28-activated polyclonal CD8 T cells cultured in the presence of IL-2 or IL-15. Recipients of 5×106 B6IL-2 (closed circles) or B6IL-15 cells (open circles) were challenged with LCMV on day 9 after transfer. (A) Viral titers were determined in the spleen 4 days after infection. Data are pooled from two independent experiments. (B) Expansion of donor T cell derived LCMV gp33- and np396-specfic cells in recipient mice after LCMV infection. Donor T cells were traced in the blood of the recipient mice using Thy1.1-specific mAb and Db-gp33 and Db-np396 tetramers. Closed (B6IL-2) and open (B6IL-15) circles represent values from individual mice.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

It is well documented that antigen-experienced T cells exist in several distinct stages of differentiation 14, 15. For adoptive T-cell therapy, it is essential to know the differentiation stage with maximum therapeutic efficacy. The ability of CD8 T cells to kill target cells or to release IFN-γ is often used to predict in vivo efficacy. However, Restifo and co-workers 8–10 made the important observation in the pmel-1 TCR-tg model that adoptively transferred CD8 T cells with full effector functions in vitro were less effective in B16 tumor cell elimination in vivo compared with less-differentiated T cells. The results reported here are well in line with these findings, although our regimen of adoptive T-cell therapy was notably different since it did not involve lymphodepletion, additional antigen boostering or cytokine treatment. In the pmel-1 transfer model, the low in vivo efficacy of pmel-1 effector cells was explained by impaired priming of the transferred T cells due to inefficient trafficking to secondary lymphoid tissues 10 and due to their terminal-differentiation stage 8.

In the transfer systems described here, P14 T cells cultured in saturate amounts of IL-2 (P14IL-2 cells) were inefficient in eliminating tumor cells or reducing LCMV titers in contrast to P14IL-15 cells that exhibited high efficacy in vivo. Moreover, the experiments revealed that P14IL-2 cells disappeared rapidly in the recipient mice after transfer whereas P14IL-15 cells persisted for prolonged time. These results fit well to the observation in patients with metastatic melanoma, which show that persistence of transferred T-cell clonotypes correlates with tumor regression 16. It is remarkable that highly cytolytic P14IL-2 cells were not able to lower B16gp33 metastases in the lung, an organ that is infiltrated by these cells immediately after transfer. This suggests that rapid tumor cell lysis by P14IL-2 cells was of minor importance for tumor cell control in our model system. Elimination of B16gp33 pulmonary metastases by activated P14 T cells requires IFN-γ but does not depend on perforin-mediated cell lysis 17. P14IL-2 cells were efficient in IFN-γ production but nevertheless failed to lower pulmonary metastases. This reinforces the view that the lack of in vivo efficacy of P14IL-2 cells was due to their short survival in vivo after transfer.

Persistence versus rapid disappearance of IL-15 versus IL-2 stimulated CD8 T cells after adoptive transfer was not a peculiarity of the P14 system since we obtained the same results with polyclonal T cells from LCMV-immune B6 mice activated by anti-CD3/CD28 antibodies. Similarly, human T cells transduced with a chimeric antigen receptor specific for CD19 were reported to be more effective in eradication of B cell tumors in SCID-Beige mice after ex vivo expansion in the presence of IL-15 18. So, why do activated CD8 T cells cultured in IL-15 survive so much better in vivo than cells kept in IL-2? Mechanical sequestration or specific retention of the blastoid P14IL-2 cells in lung and liver tissues may represent a factor that contributes to the rapid disappearance of P14IL-2 cells in the host. Nonetheless, our data also show that P14IL-15 cells survived markedly better than P14IL-2 cells after transfer into the peritoneal cavity.

Although IL-2 and IL-15 share common receptor components and signaling pathways, there are fundamental differences in the way these two cytokines act and signal 5, 7, 19, 20. IL-2 is a soluble factor produced by activated T cells 21 whereas IL-15 is complexed with the IL-15 receptor α chain (IL-15Rα) and presented on the cell surface 22–24. IL-15/IL-15Rα complexes undergo recycling without lysosomal degradation and therefore, membrane-bound IL-15 is well suited to provide continuous survival signals for receptive T cells 25–27. Moreover, signaling by IL-15 but not IL-2 has been shown to upregulate the anti-apoptotic protein Bcl-xL in anti-CD3-stimulated human T cells 18. Our experiments revealed that P14IL-2 cells were more susceptible to cytokine withdrawal in vitro than P14IL-15 cells and that IL-15 only partially protected P14IL-2 cells from rapid apoptosis in contrast to P14IL-15 cells that survived well in the presence of this cytokine. So, P14IL-2 cells may get “addicted” to high levels of IL-2 and die when they do not receive that cytokine support in vivo. In contrast, P14IL-15 cells may have access to enough IL-15 in vivo and therefore, they survive much better. High dose IL-2 treatments of the recipient mice only transiently increased the frequencies of P14IL-2 cells in the lung but failed to sustain survival of these cells in the periphery. It is well established that IL-2 treatment enhances efficacy in adoptive T-cell immunotherapy but it is important to stress that these treatment protocols are usually applied in lymphopenic hosts. In normal hosts, IL-2 treatment may be less efficient since endogenous T cells could also compete for the administered cytokine.

Adoptive transfer of Listeria monocytogenes-specific CD8 T cell lines cultured for 2–3 wk in low amounts of IL-2 and IL-7 has been demonstrated to confer protective immunity to naive recipient mice 28. Remarkably, the transferred CD8 T cells rapidly lost their effectiveness and were deleted in response to the challenge infection. In the experiments described here with IL-15-cultured CD8 T cells from P14 or LCMV-immune mice, the transferred T cells did not lose their protective and replicative ability when the recipient mice were challenged with LCMV 9 days after transfer. This discrepancy might be due to the different infection models analyzed (L. monocytogenes versus LCMV) or due to the different cytokines (IL-2/IL-7 versus IL-15) used for in vitro expansion of reactive T cells. Interestingly, anti-CD40 treatment known to induce IL-15Rα on DCs 29 rescued non-responsiveness and protective capacity of the transferred L. monocytogenes-specific CD8 T cells in the study described above 28. LCMV-specific CD8 T-cell clones have been demonstrated to mediate clearance of LCMV after adoptive transfer 30. In these experiments, the T-cell clones were transferred into pre-infected recipients and viral titers were determined 3–4 days after transfer. This is different to our experimental protocol in which the in vitro cultured T cells were first “parked” in the recipient mice for 9 days before LCMV infection.

Our data also showed that the period of time of in vitro culture with IL-2 played a crucial role for the in vivo survival of P14IL-2 cells after adoptive transfer. These data are well in line with a previous report by Malek and co-workers in the OT-1 model 13. These data may also help to reconcile the discrepancy between our data and a previous report by Weninger et al. 12 in which P14IL-2 cells could be recovered from the blood 24 h after transfer. In the latter study, activated P14 T cells were cultured for 6–8 days in IL-2 before transfer. As illustrated by the present data, a 2-day difference of in vitro culture may strongly influence cell survival after transfer also in the P14 system. Furthermore, subtle differences in tissue culture conditions including cell culture media or fetal calf serum (FCS) batches may additionally influence the extent of effector T-cell differentiation in vitro and the resulting in vivo survival after adoptive transfer.

The generation of substantial numbers of antigen-specific T cells is a prerequisite for successful adoptive immunotherapy. Short-term culturing of T cells in IL-2 is only possible when sufficient numbers of antigen-specific T cells are already available at the beginning of the in vitro expansion. In model systems, this can be achieved by the use of lymphocytes from TCR transgenic mice. Under more physiological conditions, it is unlikely that short-term cultures will allow the generation of sufficient numbers of antigen-specific T cells. Therefore, it is important to optimize culture conditions for long-term in vitro expansion of T cells. Our data revealed that P14 T cells cultured in low amounts of IL-2 exhibited improved survival and efficient anti-tumor and anti-viral activity after transfer. However, lowering the IL-2 concentration also decreased the total cell yield of the T-cell cultures. Thus, a decrease in IL-2 signaling reduced effector cell differentiation but also impaired in vitro expansion of the activated T cells. In contrast, IL-15 did not induce terminal effector cell differentiation but still allowed the generation of substantial numbers of T cells in vitro.

In conclusion, the present report supports the use of IL-15 for the in vitro expansion of human CD8 T cells also for clinical settings that do not involve lymphodepletion, antigen boostering or exogenous IL-2 administration. In addition, the results of the present study might be helpful for the current efforts that utilize genetically engineered T cells 31–33 for adoptive immunotherapy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

Mice

C57BL/6 (B6) mice were obtained from Harlan Winkelmann (Borchen, Germany). Thy1.1+ P14 TCR-tg mice (B6; D2-Tg(TcrLCMV)318Sdz/JDvsJ) specific for aa 33-41 (gp33 epitope) of the LCMV glycoprotein 34 and B6.Thy1.1 mice (B6.PL-Thy1a) were bred locally. Female or male mice were used at 8–20 wk of age. Mice were bred and kept under specific pathogen-free conditions. All animal experimental protocols used in this study were approved by the Regierungspräsidium Freiburg.

Melanoma lung metastases model

B16.F10 melanoma cells expressing the gp33 epitope (=B16gp33) 35 were cultured in DMEM high glucose supplemented with 10% FCS, 2 mM L-glutamine, streptomycin, penicillin (100 U/mL) and were kept under G418 selection (0.6 mg/mL) (all from Life Technologies, Gaithersburg, MD, USA). Experimental pulmonary metastases were induced by i.v. injection of 106 B16gp33 cells in 500 µL PBS via the lateral tail vein. After 2 days, P14IL-2 or P14IL-15 cells (5×106 in 0.5 mL PBS) were i.v. injected. The number of macroscopically visible metastases on the lung surface was determined 2 wk after tumor cell transfer.

Virus

The LCMV strain WE used in this study was originally obtained from Prof. Lehmann-Grube (Heinrich-Pette-Institute Hamburg, Germany). It was propagated on L929 fibroblasts and quantified in a virus plaque assay 36. Mice were infected with 200 PFU of LCMV-WE i.v. and virus titers in the spleen were determined 4 days after infection.

Generation of P14IL-2 and P14IL-15 cells and adoptive transfers

Spleen cells (2×106/mL) from P14 TCR-tg mice were stimulated for 2 days in 24-well plates in 1 mL culture medium (Iscove's modified Dulbecco's medium supplemented with 10% FCS, penicillin/streptomycin, 0.001 M β-mercaptoethanol) in the presence of 10−6 M gp33 peptide (KAVYNFATM, Neosystem). Afterwards, the cells were harvested and cultured in 24-well plates at 0.5×106 and 1×106 cells per well in fresh medium containing the indicated concentration of either human IL-2 (Proleukin, Novartis Pharma, Basel) or human IL-15 (Research Diagnostics, Flanders, NJ, USA), respectively. Every second or third day, the cells were harvested, counted and re-cultured in fresh medium supplemented with cytokines. P14IL-2 and P14IL-15 cells were labeled with CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl) amino)tetramethylrhodamine, Molecular Probes, Eugene, OR, USA) and CFSE (Molecular Probes), respectively, according to the instruction of the manufacturer. All experiments were done using cells cultured for 9 days and the cells (5×106) were injected i.v. in 0.5 mL PBS. Survival of P14 T cells in the peritoneal cavity was determined after i.p. injection of 5×106 cells in 0.5 mL PBS. High dose IL-2 treatment was performed by i.p. injection of recombinant human IL-2 (Proleukin, Novartis Pharma, 600.000 IU/36 µg per dose), administered twice daily at days 0, 1 and 2 after T-cell transfer.

Polyclonal LCMV-specific T cells

Spleen cells (106/mL) from LCMV immune (week 5–8 post infection) B6.Thy1.1 mice were stimulated for 2 days with magnetic beads coated with anti-CD3/CD28 antibodies (Dynabeads Mouse CD3/CD28 T-cell expander, Dynal, Invitrogen, Karlsruhe, Germany) according to the instruction of the manufacturer. After removal of the beads, the cells were cultured in medium containing IL-2 (20 ng/mL) or IL-15 (50 ng/mL) for additional 7 days and injected i.v. as described above.

Flow cytometry

To obtain single cell suspensions of spleen and inguinal lymph nodes, the organs were smashed, washed and filtrated through a 100 µm cell strainer (BD bioscience). To isolate lymphocytes from liver and lung, organs were cut into small pieces and digested in PBS containing 0.1% collagenase, 0.01% hyaluronidase and 0.002% DNase I (all from Sigma-Aldrich, Steinheim, Germany) for 30 min at 37°C. Afterwards, the organs were smashed and lymphocytes were purified by Ficoll gradient centrifugation (Ficoll-PaquePlus; Amersham Biosciences, Uppsala, Sweden). Staining was performed in PBS containing 2% FCS and 0.1% NaN3. The following mAb were used: FITC-labeled anti-Thy1.1, anti-CD8, anti-CD62L, anti-CD25, PE-labeled anti-CD62L, anti-CD122, anti-TCR Vα2 (all from BD PharMingen), PE-Cy5.5-labeled anti-CD8 (Caltag) and APC-labeled anti-CD8 (BD PharMingen). CD132 expression was determined by purified anti-CD132 mAb (BD PharMingen) followed by goat-anti-rat IgG-PE (Caltag). IL-15Rα expression was analyzed by staining with biotinylated goat-anti-mouse IL-15Rα antibody (R&D Systems) followed by streptavidin-APC (BD PharMingen). CCR7 expression was determined by using a chimeric CCL19-Ig fusion protein. Apoptotic cells were analyzed by using Annexin V-FITC apoptosis detection kit (BD PharMingen) according to the instructions of the manufacturer. LCMV-specific CD8 T cells were determined using PE-conjugated gp33–41− and np396–404 H-2Db tetramers. Before analysis of PBL, red blood cells were lysed using FACS-Lysing Solution (BD PharMingen). Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) using CELLquest software.

Cytotoxicity and IFN-γ assays

The cytolytic activity of P14IL-15 and P14IL-2 cells was determined in standard 5 h 51Cr-release assays. Briefly, target cells (∼5×106) were labeled with 250 µCi 51Cr in ∼0.5 mL culture medium at 37°C for 2 h. For peptide loading, 10-6 M gp33 peptide or control Db-binding adenovirus peptide 234–243 (E1A peptide, SGPSNTPPEI) was added. L1210 cells over-expressing CD95/Fas have been described previously 37. The assays were performed in 96-well round bottom plates using 104 target cells per well in a total volume of 200 µL. To determine IFN-γ production, P14IL-15 and P14IL-2 cells were cultured for 4 h in 24-well plates (2×106 cells per well) together with B6 spleen cells (2×106per well), pre-coated (1 h, 37°C, 10−6 M) with gp33 peptide. Afterwards, cells were surface stained with anti-CD8-APC and anti-Thy1.1-FITC mAb, fixed and permeabilized using Cytofix/Cytoperm solution (BD PharMingen) and stained intracellularly with PE-conjugated anti-IFN-γ mAb (BD PharMingen) according to the instruction of the manufacturer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. References

We thank Peter Aichele and Andreas Diefenbach for their critical comments on the manuscript, Norma Bethke, Rainer Bronner, Christian Herr and Sonja Wagenknecht for animal husbandry, and Juergen Brandel for help with image processing and artwork. This work was supported by the Deutsche Forschungsgemeinschaft DFG (PI 295/5-2 to H.P.).

Conflict of interest: The authors declare no commercial or financial conflict of interest.

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  4. Results
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  6. Materials and methods
  7. Acknowledgements
  8. References
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