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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Immunology [J. L., R. O., H. K., E. C.] and Obstetrics and Gynecology [R. L. G.] and Mayo Graduate School [R. K., E. C.], Mayo Clinic, Rochester, Minnesota 55905
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which was detected on CD8+ CTLs but not on CD4+ helper T cells, suggests that this receptor subunit somehow participates in the transduction of the mitogenic signals of IL-15. The present findings have practical implications for the propagation of antigen-specific T-cell lines in vitro and could be useful for expansion of therapeutic T cells for adoptive transfer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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50 IU/ml) for 3–4 weeks without much additional increase in cell numbers but retaining their effector function. Among the various factors that seem to control the expansion and viability of antigen-activated T lymphocytes, IL-2 and IL-15 are perhaps the most extensively studied. Although IL-2 and IL-15 share two of three receptor subunits on the T-lymphocyte surface, their biological function can be quite different depending on the activation state and the specific subset of T lymphocytes that is targeted by these lymphokines (4 , 5) . Although both of these lymphokines appear to function as T-cell growth and survival factors, in some circumstances IL-2 can also be involved in the down-regulation of T-cell responses mediated through mechanisms such as activation-induced cell death. On the other hand, IL-15 has been regarded by many as an antiapoptotic lymphokine that plays a critical role for the survival of memory CD8+ CTLs (6, 7, 8) . Most significantly, in the presence of IL-15, tolerizing TCR stimuli result in a state of reversible T-cell anergy instead of apoptosis (9) . In addition, IL-15 has also been reported to prevent in vitro apoptosis of T cells from patients after autologous progenitor cell transplantation (10) . However, in HIV-infected individuals, IL-15 failed to function as an antiapoptotic factor in nave and memory CD8+ T cells, suggesting that the effects of this lymphokine may differ in some diseases affecting the immune system (11) . In view of the above, we decided to evaluate the role of IL-15 in the maintenance of antigen-specific CTLs in culture and to determine whether these cells would retain their effector function or whether they would switch into a memory (nonlytic) phenotype, as it has been reported to occur with mouse CTLs (12) . The data presented herein demonstrate that human CD8+ CTLs expand in an antigen-independent fashion and retain their effector phenotype for up to 60 days, when IL-15 or high concentrations of IL-2 (1000 IU/ml) but not low concentrations of IL-2 (50 IU/ml) are included in the tissue culture medium. The effect of IL-15 on CD8+ T cells appears to be product of an increase in the rate of proliferation concomitant with a decrease in the numbers of cells undergoing apoptosis. In contrast, IL-15 and IL-2 (at either concentration) failed to stimulate the antigen-independent expansion of CD4+ T cells, indicating that signaling and survival pathways triggered by the IL-2R and IL-15R differ between these lymphocyte subsets.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Assessment of Cell Expansion.
Antigen-specific human CD4+ and CD8+ T-cell lines or clones were induced from healthy donors using peptide-pulsed autologous DCs as described previously (3
, 13, 14, 15)
. The DCs used to generate the antigen-specific T-cell lines and clones were prepared from adherent monocytes that were incubated in tissue culture for 7 days in the presence of granulocyte macrophage colony-stimulating factor (50 ng/ml) and IL-4 (1000 IU/ml), without additional maturation (3)
. CTL clone X283 specifically recognized HLA-A2-restricted peptide gp100209 (ITDQVPFSV) derived from melanoma gp100 antigen (16)
, and CTL clone PSMA1126.14 was specific for HLA-A2-restricted peptide PSMA469 (LMYSLVHNL) derived from PSMA.4
T-helper clones 10B1 and 3F12 recognize peptide gp10074 (GPTLIGANASFSIALN) and peptide gp100576 (SLAVVSTQLIMPGQE), respectively, both in the context of HLA-DR7 (15)
. All of the human T cells were maintained in tissue culture using cytokine-supplemented complete RPMI medium (RPMI 1640 plus 10% fetal bovine serum, L-glutamine, nonessential amino acids, sodium pyruvate, and lentamicin). Fresh medium supplemented with cytokines was added every 2 or 3 days. For reactivation, the T cells were stimulated with 30 ng/ml human anti-CD3 (Orthoclone OKT3; Ortho Biotech, Inc., Raritan, NJ) in the presence of irradiated feeder cells and sufficient amount of IL-2 for 2 weeks before additional use (3)
. Effector T cells derived from CBMCs were generated by stimulation with 1 µg/ml OKT3 for a week and then purifying the CD8+ T cells using antibody-coated magnetic beads (Miltenyi Biotec, Auburn, CA). A CTL clone specific for peptide gp100209, derived from TILs from a melanoma patient was kindly provided by Dr. Franco Marincola (Surgery Branch, NCI, NIH). These cells were maintained in complete medium containing 1000 IU IL-2/ml. The T2 cell line (HLA-A2+) was kept in complete RPMI medium and used as targets for cytotoxicity assays. To determine survival and expansion of T cells, the cultures were initiated at 5 x 105 cells/ml in complete RPMI medium with various cytokines. Every 3–5 days, the numbers of viable cells were estimated using trypan blue exclusion, and the cells were resuspended at the original cell concentration in fresh media containing the corresponding cytokine.
Cytotoxicity Assays and Target Cell Lines.
Effector function of CD8+ T cells was evaluated at several time points by measuring the cytolytic activity using a standard 4–6 h chromium release assay as described previously (3)
. Approximately, 1 x 106 peptide-pulsed T2 cells were labeled with 100 µCi 51Cr for 1.5 h and, after extensive washing, were used as targets in the cytotoxicity assays at various E:T ratios. Target cell lysis was determined by measuring the amount of radioactivity released by the lysed target cells into the supernatant using a scintillation counter. The percentage of specific lysis was calculated by the formula ([cpm of the test sample - cpm of spontaneous release]/[cpm of the maximal release - cpm of spontaneous release] x 100). The transporter associated with antigen processing-deficient, HLA-A2+, T2 human cell line (17)
was used as peptide-pulsed targets for the HLA-A2-restricted CTLs. The HLA-A2+, gp100+ human melanoma, and 624mel was provided by Dr. Steven A. Rosenberg (Surgery Branch, NCI, NIH). Both of these cell lines were maintained complete RPMI medium.
Cell Proliferation Assays.
The function of CD4+ T cells was assessed by the capacity of these cells to respond to antigen by the incorporation of [3H]thymidine into DNA, which correlates with cell proliferation, as described previously (14
, 15)
. Briefly, T cells (2 x 104 /well) were mixed with irradiated autologous peripheral blood mononuclear cell (1 x 105/well) in the presence (and absence) of 3 µg/ml of the corresponding peptide in 96-well culture plates. The cultures were incubated at 37°C in a 5% CO2 incubator for 72 h and during the last 16 h, each well was pulsed with 0.5 µCi/well of [3H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ). The radioactivity incorporated into DNA, which correlates with cell proliferation, was measured in a liquid scintillation counter (TopCount; Packard Instruments) after harvesting the cell cultures onto glass fiber filters.
Antibodies and Flow Cytometry.
Monoclonal antibody M161, specific for the human IL-15R chain, was originally developed by Immunex Corp. (Seattle, WA) and was kindly provided by Genmab A/S (Copenhagen, Denmark). The perforin antibody reagent set, phycoerythrin-conjugated anti-CD25 (IL-2R), CD122 (IL-2Rß), CD132 (IL-2R
), and isotype-matched IgG controls were all purchased from BD PharMingen (San Diego, CA). Expression of IL-2R and IL-15R chains on T cells was determined by fluorescent antibody staining and flow cytometry using a FACScan machine and CellQuest software (Becton Dickinson, Biosciences, San Jose, CA). Cell cycle analysis was performed by staining permeabilized T cells with propidium iodide (PharMingen) and subsequently running the samples in the FACScan machine and analyzing the results with the ModFit LT v2.0 software (Verity Software House, Topsham, ME).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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700-fold expansion, the cells were harvested, washed, resuspended in fresh media containing either IL-2 or IL-15, and the cell numbers were monitored 3–4 times/week. As routinely observed in our laboratory, cells resuspended in IL-2 did not expand any further but were able to maintain their viability for 2 weeks (Fig. 1A)
. Interestingly, the CTLs resuspended in IL-15 (10 ng/ml) continued to expand at an accelerated rate for 60 days, increasing 120-fold in cell number (Fig. 1A)
. In separate experiments, we observed that CTL cultures in IL-4, IL-7, or IL-10 (10 ng/ml each) or in the absence of lymphokines gradually decreased in cell numbers and failed to maintain their viability (data not presented). Most importantly, the cytolytic activity of the CTLs did not decrease after 60 days in culture with IL-15 (Fig. 1B)
, and the levels of intracellular perforin remained constant (Fig. 1C)
, indicating that the effector phenotype of these cells was stable. Moreover, the quality of the cytolytic activity of these CTLs, as assessed by peptide titration curves, remained constant after 60 days in IL-15 (Fig. 1D)
. The functional characterization of the CTLs maintained in IL-2 could not be assessed at day 60 because by that time these cells had all died, but these cells retain their effector function during the first 2 weeks (data not shown).
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. Again, the CTLs maintained in IL-15 expanded 100-fold, whereas the cells in IL-2 had a limited proliferative potential (Fig. 2A)
. The cytolytic activity of the cells grown in either IL-2 or IL-15 was preserved while the cells retained viability (Fig. 2B)
and the expression of intracellular perforin remained high after 60 days in culture in IL-15 (data not shown). Most significantly, the quality of the cytolytic activity of these CTLs did not decrease after 30 days in culture in IL-15, as determined by the capacity of the CTLs to kill melanoma tumor cells (Fig. 2D)
and in peptide titration curves (Fig. 2C)
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40 days after the initiation of the experiments (Figs. 1A
and 2A
). From that time on, the cultures maintained constant cell numbers for 20 additional days and then the cell numbers slowly decreased until the viability was lost (day 80). However, when the CTLs that had reached stationary growth in IL-15 (60 days) were stimulated with anti-CD3 antibodies, APC and IL-2 (50 units/ml), they expanded 300–500-fold during a period of 2 weeks, and if placed in IL-15 after this time period, they expanded an additional 100-fold in a matter of 30 days (data not presented). Throughout this time, the CTLs continued to maintain their cytolytic function (data not shown).
The results presented above were obtained using antigen-specific CTLs that were generated in vitro from normal individuals using peptide-pulsed DCs (3)
. To determine whether these observations would also be applicable to CTL derived from cancer patients, we compared the capacity of IL-2 and IL-15 to expand a CTL clone derived from melanoma TILs. In these experiments, we also included a high-concentration IL-2 condition because TILs have been reported to require high concentrations of this lymphokine to grow in vitro (18
, 19)
. The results observed with the melanoma patient-derived CTLs were very similar to those obtained with the peptide-pulsed DC-generated CTLs. The data presented in Fig. 3A
indicate that IL-15 or the high concentration of IL-2 (120 ng/ml or 1000 units/ml) was equally effective in stimulating the melanoma patient’s CTLs to proliferate in vitro up to 100-fold in the absence of antigen, and the cell viability was maintained constant for at least 35 days. On the other hand, a low-dose IL-2 (50 units/ml) induced a much lower degree of proliferation (10-fold), and the cell viability declined from day 15 onwards. Most significantly, the cytotoxic activity of the TIL-derived CTLs that were maintained in either IL-15 or high-dose IL-2 for 15 days remained high (Fig. 3B)
. Moreover, after 30 days in culture in IL-15, the TIL continued to recognize peptide and tumor cells in a high efficient manner (Fig. 3, C and D)
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120–100-fold in a period of 40–50 days and then proliferation appeared to stop. Because the CTLs used in these experiments had all been continuously propagated in cell culture for 3–6 months, the possibility existed that the cells ceased to expand because they had reached a senescence state. However, as mentioned above, TCR stimulation was able to reinitiate the proliferation of CTLs that had reached stationary growth in IL-15. To determine whether CD8+ T cells with a short proliferation history would be able to expand in IL-15 for longer periods of time than long-term cultured CTLs, we evaluated the capacity of CD8+ T cells obtained from CBMCs to proliferate and survive in IL-15 and IL-2. The growth curves presented in Fig. 4
, using purified short-term TCR-activated CD8+ T cells that were isolated from CBMC of two separate donors, show very similar patterns to those obtained using long-term cultured CTLs. Once more, the cells maintained in IL-15 expanded close to 120-fold in a matter of 40 days, whereas the cells in IL-2 had a more limited expansion potential. The small, but significant (10-fold) expansion of these cells in IL-2, could be because these cells remained in a highly activated state because they had been stimulated with anti-CD3 antibody only 7 days before the initiation of the experiment. These results suggest that the cell expansion potential after TCR stimulation of CTLs incubated in IL-15 appears to be independent of the proliferative past of the cell cultures.
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, both T cell clones exhibited a limited expansion potential (3-fold max) in the presence of IL-15 or IL-2. Nonetheless, both IL-2 and IL-15 were able to preserve the viability of the cell cultures up to day 58 when this experiment was terminated (Fig. 5, A and B)
. Conversely, the CD4+ T-helper cell cultures that were maintained in the absence of exogenous lymphokines rapidly decreased in cell numbers, and most of the cells were dead by day 15. The function of the CD4+ T cells cultured in either IL-2 or IL-15 was preserved as assessed by antigen-induced proliferation assays (Fig. 5, C and D)
. These responses were slightly higher on the CD4+ T cells maintained in IL-15 as compared with the cells grown in the presence of IL-2. The variability in the proliferative responses observed between different time points, which in some cases appeared to increase (Fig. 5C)
and in other cases decrease (Fig. 5D)
, could be because of variances in the proliferation assays because these were performed in different days. Nevertheless, at each individual time point, the responses of the T cells were very similar regardless of whether they were maintained in IL-2 or IL-15. The overall results indicate that IL-15 displays different effects in the expansion of CD8+ and CD4+ T cells as previously suggested (6)
. Nonetheless, it is evident that IL-2 and IL-15 can serve as viability preserving factors for both CD4+ and CD8+ T cells.
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. Furthermore, the proportion of cells having DNA debris (which is an indication of apoptotic cell death) that was evident in the cultures maintained in IL-15 was significantly lower than in the IL-2 cultures. Similar results were obtained with other T-cell lines and clones (data not presented). These results indicate that the expansion of CD8+ T cells in IL-15 is the product of an increase rate of proliferation and decrease in the degree of cell apoptosis.
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), each cytokine receptor has a unique chain (20, 21, 22)
. We measured the expression of the various components of the IL-2 and IL-15 receptors on the surface of recently (2-week) TCR-stimulated T-cell clones by flow cytometric analysis. The results indicate that the IL-15R chain was detected in only CD8+ T cells (Fig. 7A)
and not on the CD4+ T cells (Fig. 7B)
. Although both CD4+ and CD8+ T-cell subsets expressed the IL-2R chain, the levels of expression on CD4+ cells were substantially lower than those observed on the CD8+ T cells (Fig. 7, C and D)
. Lastly, no apparent differences were detected in the levels of expression of the shared ß and chains (Fig. 7, E–H)
. Interestingly, when CD8+ T cells were resuspended in medium containing IL-15, the expression of the IL-15R was lost after a few days, but the expression of the ß and chains remained unaltered (data not shown), suggesting that the IL-15R chain may be down-regulated by IL-15.
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, which is evident on these cells but not on CD4+ T lymphocytes. These results suggest that IL-15 may directly transmit a proliferative signal to the T cell by binding to IL-15R. Alternatively, IL-15R, which has a very high affinity for IL-15 (1011) may stabilize the binding of lymphokine to the ß complex, which then transmits the proliferative signal. The latter would explain why high concentrations of IL-2 could mimic the effects of IL-15 on TIL-derived CTLs (Fig. 3A)
. This scenario would predict that CD4+ T cells should proliferate to high concentrations of IL-2 because the levels of expression of their IL-2/15ß and IL-2/15 chains were similar to those found in CD8+ T cells (Fig. 7, E–H)
. On the other hand, if CD4+ T cells fail to proliferate to high concentrations of IL-2, this would suggest that IL-15R does play a role in the transduction of the proliferative signal in CD8+ T cells. To examine these possibilities, we measured the capacity of CD4+ T cells to proliferate in an antigen-independent way to high concentrations of IL-2 (1000 IU/ml). The results presented in Fig. 8
, showing that CD8+ but not CD4+ T cells proliferated to the high-dose IL-2 (and to IL-15), indicate that IL-15R actively participates in the proliferative response of CD8+ T cells in the absence of antigen.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
and 6
). As reported in the mouse system, human CD4+ T cells did not respond to IL-15 in the same manner as CD8+ T cells did (Figs. 5
and 8
). Our data also shows that although IL-15 was unable to induce the expansion of CD4+ T cells, it was capable of functioning as a survival factor in a similar way as IL-2 (Fig. 5)
. In contrast to mouse CD8+ memory cells where antigen-independent proliferation presumably continues throughout the animal’s life, the human CD8+ effector cells ceased to divide in culture after 30–40 days and their viability declined unless they received TCR activation signals, which may be required for the re-expression of IL-15R (6)
. Thus, it is possible that effector and memory CD8+ T cells may respond differently to IL-15. Alternatively, it is possible that memory CD8+ T cells continue to express IL-15R without the need of TCR stimulation for much longer periods of time than effector CTLs. Another possible explanation for the differences observed between the in vivo mouse and the in vitro human systems is that in vivo T cells are likely to receive additional signals from other cells or the extracellular matrix, which help to maintain their responsiveness to IL-15.
The expansion of CD8+ T cells observed in IL-15 and not with IL-2 appears to be derived from both an increase in the rate of cell proliferation and a decrease in cell death (Fig. 6)
. The mechanism by which IL-15 triggers antigen-independent cell proliferation in CD8+ T cells, but not in CD4+ T cells, may be complex. Because the surface expression of IL-15R in mouse memory CD8+ T cells is undetectable, and the levels of IL-2/15Rß are much higher (10–30-fold) than those observed on memory CD4+ T cells, it has been proposed that IL-15 triggers cell division in these cells through the IL-2/15R ß receptor complex (6)
. On the other hand, the presence of the IL-15R chain on human CD8+ CTLs (Fig. 7A)
, which alone can bind IL-15 with a Ka 100-fold higher than the IL-2/15Rß complex (25)
, suggests that the proliferative signals of IL-15 on human CTLs could be mediated via the IL-15R chain. This possibility is reinforced by the observations that effector CD4+ human T cells, which did not express the IL-15R chain (Fig. 7)
, failed to proliferate in the presence of this lymphokine (Fig. 5, A and B)
. Although another possible explanation for these results is that IL-15R simply facilitates the binding of the lymphokine to the IL-2/15ß complex, which is then responsible for signal transduction for the proliferative response. However, if this were the case, high concentrations of IL-2 should be able to trigger the proliferation of the T cells because this lymphokine binds to the ß complex with a Ka of 109 M. Indeed, IL-2 at 1000 units/ml (6.8 x 10-9 M) mimicked the effect of IL-15 in CD8+ T cells, triggering their antigen-independent proliferative response. Nevertheless, the same high concentration of IL-2 did not have an effect on CD4+ T cells, although these cells expressed similar levels of IL-2/15Rß and IL-2/15R. Thus, under this scenario, one would have to propose that differences existing between CD4+ and CD8+ T cells are in the capacity of their IL-2/15ß complexes to transduce a proliferative signal. In other studies (26)
, it was reported that IL-15 was able to activate memory CD4+ T cells in addition to naïve and memory CD8+ T cells, as determined by increase in the expression of the CD69 marker and the synthesis of DNA measured by [3H]thymidine incorporation. However, these authors used much higher concentrations of IL-15 (100 ng/ml) than those used in the present studies and did not assess the long-term effects of IL-15 or the actual expansion of the cell cultures. We have observed that T-cell activation and even [3H]thymidine incorporation into DNA do not necessarily imply that the cells will be able to successfully proliferate and expand in cell numbers, because in some instances the cells undergo apoptosis.4
The present findings suggest that in addition to maintaining CD8+ T cell memory in vivo (6, 7, 8 , 12 , 24) , IL-15 may also help to enhance and prolong the effector function of CTLs. It is possible that once a pathogen has been apparently eliminated by the immune system, some of the effector cells may remain functional and even continue to expand in the peripheral tissues for some period of time as a safeguard against pathogen resurgence. In addition to the possible physiological role that IL-15 plays on effector CD8+ T-cell responses, our results bear some practical implications for the maintenance and propagation of antigen-specific T-cell lines in vitro. The use of IL-15 should enable researchers to grow CTLs for longer periods of time without loss of effector function while avoiding the cumbersome process of periodic antigen restimulation. This technique could also be useful for the expansion of therapeutic CTLs to be used for adoptive transfers.
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FOOTNOTES |
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1 Supported by NIH Grants R01CA80782, R01CA82677, P50CA91956, and RR-00585. 
2 To whom requests for reprints should be addressed, at Department of Immunology, GU421A, Mayo Clinic, Rochester, MN 55905. Phone: (507) 284-0124; Fax: (507) 266-5255; E-mail: celis.esteban{at}mayo.edu
3 The abbreviations used are: CTL, cytotoxic T lymphocyte; TCR, T-cell receptor; APC, antigen-presenting cell; DC, dendritic cell; IL, interleukin; IL-2R, IL-2 receptor; IL-15R, IL-15 receptor; PSMA, prostate-specific membrane antigen; CBMC, cord blood mononuclear cell; TIL, tumor-infiltrating lymphocyte.
4 E. Celis, unpublished results.
Received 2/25/02; revised 8/ 1/02; accepted 8/ 5/02.
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) or 10 ng/ml IL-15 (•), and numbers of viable cells were determined at various time points. B, antigen-mediated cytotoxicity of CTLs maintained in IL-15 against PSMA469 peptide-pulsed T2 cells was determined on day 0 (
) and on day 60 (
). No significant cytotoxicity was observed against T2 targets cells pulsed with a control irrelevant peptide (day 0: 
; day 60: 
). C, intracellular perforin content of CTLs grown in IL-15 was measured by flow cytometry with a perforin-specific antibody on day 0 (thick line) and day 60 (thin line). The shaded area represents staining using an antibody isotype control, and the numbers above each curve correspond to the mean fluorescence intensity. D, peptide titration curve of CTLs that were incubated for 0 days (
