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Immunology. Jun 2009; 127(2): 206–215.
PMCID: PMC2691786

Interleukin 21 up-regulates perforin-mediated cytotoxic activity of human intra-epithelial lymphocytes

Abstract

Human intra-epithelial lymphocytes (IELs) are predominantly T-cell receptor-αβ+ (TCR-αβ+) CD8+ CD45RO+ memory T cells located between intestinal epithelial cells. They respond to a greater extent to stimulation with interleukin (IL)-15 than to CD3/TCR triggering, suggesting that they react to the cytokine milieu in their local environment rather than to cognate antigen. A newly described member of the γc cytokine family, IL-21, has potent antitumor effects. As IELs resemble lymphocytes infiltrating neoplastic lesions, their response to IL-21 may be relevant in vivo. Here, IL-21 was shown to increase perforin-mediated cytotoxicity and serine esterase release by IELs. This IL-21-mediated up-regulation occurred without changes in IEL survival or cell division. Interestingly, the effects of IL-21 occurred without increased phosphorylation of signal transducer and activator of transcription (STAT)1, STAT3, STAT4, STAT5, extracellular signal-regulated kinase (ERK), or p38. IL-21 had no effect on Fas ligand (FL)-or tumour necrosis factor-α (TNF-α)-mediated cytotoxicity, but it down-regulated IL-15-stimulated expression of CD25 and CD94, indicating that it has both positive and negative actions. This functional profile is unique to human IELs, emphasizing that they are a distinct compartment of lymphocytes and that IL-21 may promote their role in tumour immunosurveillance.

Keywords: cytotoxicity, Fas ligand, interleukin-21, intra-epithelial lymphocytes, perforin, tumour necrosis factor

Introduction

Human intra-epithelial lymphocytes (IELs) are predominantly T-cell receptor-αβ+ (TCR-αβ+) CD8+ CD44+ CD45RO+ CD62low memory T cells located between intestinal epithelial cells (ECs).1 Murine IELs differ in phenotype and function, with TCR-γδ T cells comprising up to 60% of the total depending upon the age of the animal and their exposure to bacteria. Human IELs, instead, phenotypically resemble lymphocytes infiltrating neoplastic lesions, with expression of CD44, CD45RO, CD69, CD122 and CD103 (αEβ7) but with few CD28+ or CD56+ lymphocytes.2,3 They are in a prime location to contribute to the early host defense against dysplastic ECs and early colon cancer.

IELs carry out spontaneous cytotoxic (SC) activity and lymphokine-activated killer (LAK) activity against colon cancer cells, suggesting that they identify and destroy dysplastic and frankly malignant tumour cells.4,5 Binding of NKG2D to its ligands, UL16-binding protein (ULBP) and major histocompatibility complex (MHC) class I chain-related (MIC) proteins found on colon cancer cells, triggers Fas ligand (FL) production by IELs, which, in combination with tumour necrosis factor-α (TNF-α), is responsible for SC activity.6 Perforin is not involved, thereby distinguishing SC activity from natural killer (NK) activity. LAK activity is triggered by interleukin (IL)-15, which is presented on the surface of antigen-presenting cells by the IL-15α receptor subunit.7 LAK activity, in contrast to SC activity, is mediated by FL and perforin, but not by TNF-α.8 IL-15 increases NKG2D and CD94 expression on IELs, and this increase in expression is independent of TCR ligation.9,10 CD94 is coupled with either inhibitory or activating NKG2 molecules; the complex recognizes the non-classical, non-polymorphic molecule, HLA-E, found on colon cancer cells. The CD94–NKG2 complex may be involved in the final lytic events. IEL cytotoxicity is normally down-regulated by low NKG2D expression on unstimulated IELs and by diminished levels of HLA-E on intestinal epithelial cells.1

IL-21 is a newly described member of cytokines utilizing the common γ-chain (CD132) receptor subunit.11 It is produced by activated CD4+ T cells and NK T cells as well as by human intestinal CD3+ lamina propria T lymphocytes (LPLs) particularly in Crohn's disease.1215 The production of IL-21 by LPLs may influence IEL function. IL-21 enhances the expression of IL-21 RNA and protein in peripheral blood CD3+ T cells and LPLs.15

The IL-21 receptor is expressed on T cells, B cells, dendritic cells, NK cells and particularly on antigen-specific CD8+ T lymphocytes.16,17 IL-21 promotes the growth of central memory (CD44high CD62hi, CCR7+) T cells, a CD45RO+ CD28high CD8+ T-cell subset and effector memory (CD44high 62Llow) CD8+ T cells, the last resembling the phenotype of human IELs.1,1820 Other studies, in contrast, show that IL-21 increases the proliferation of naïve, but not of memory, T cells 12 and counteracts the expansion of NK cells and CD44hi CD8+ T cells.21,22 IL-21 promotes the apoptosis of NK and T cells, but not of NK T cells or CD8+ T cells.13,16,18,21,22 It increases IL-15-induced T-cell mRNA synthesis for interferon-γ (IFN-γ) and phosphorylation of signal transducer and activator of transcription (STAT)1, STAT3 and STAT4 at the regulatory sites of the IFN-γ gene in T cells.23 Alone, IL-21 phosphorylates STAT1 and STAT3 in a human T-cell leukaemia line.11

IL-21 augments NK cell activity, cytotoxic T lymphocyte (CTL) activity and memory CTL responses to tumour rechallenge in many systems.12,18,20,21,2428 It can augment or reduce NKG2D-dependent cytolysis 23,24 and increase or decrease CD94–NKG2A expression.22,29,30 IL-21 up-regulates the expression of perforin and granzyme B in most 13,19,22,24,26,28,29 but not all 31 studies. Taken together, most reports show that IL-21 is an up-regulator of cytotoxic activity mediated by perforin.

The present study analyzed the effects of IL-21 on human IELs, either alone or with IL-15. In light of the importance of IL-15 in IEL function 5 and the anti-tumour effects of IL-21, the role of IL-21 in IEL biology is of particular interest. This study showed that IL-21 increases serine esterase release by IELs and redirects lysis against P815 cells.

Materials and methods

Isolation of lymphocytes

Peripheral blood lymphocytes (PBLs) were isolated by Ficoll density-gradient centrifugation. IELs were separated from jejunal mucosa from otherwise healthy patients undergoing gastric bypass operations for morbid obesity after obtaining informed consent as described in detail previously.46 Briefly, minced mucosa was treated in a shaking water bath (37°) for 60 min with 1 mm dithiothreitol (Sigma-Aldrich, St Louis, MO) diluted in RPMI-1640 containing 10% fetal calf serum, glutamine and antibiotic–antimycotic solution (complete medium). The mucosa was then treated with 0·75 mm ethylenediamine tetraacetic acid (EDTA; Sigma-Aldrich) and washed every 45 min with Hanks’ balanced salt solution (Biowhittaker, Walkersville, MD), pH 7, for three cycles. All cells were collected and separated by a Percoll density gradient (Pharmacia, Piscataway, NJ). The IELs, which layered over the 40% solution, were recovered. After isolation, the purity, determined by immunofluorescence (IF) with anti-CD45 IgG immunoglobulin (R&D Systems, Minneapolis, MN), was over 90%.

Functional analyses

IELs (2 × 106/ml) were stimulated in complete medium supplemented with or without anti-CD3 IgG immunoglobulin (50 ng/ml) in addition to IL-15, IL-21, or the combination of both (1–100 ng/ml; all from R&D Systems). The following functions were then analyzed.

Determination of numbers of viable IELs using the MTT assay. IELs were incubated with IL-15 and/or IL-21 (10 ng/ml) for 1–3 days. 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, final concentration of 0·5 mg/ml; Sigma-Aldrich) was added each day and the cells were incubated for 6 hr at 37°. Colour development was initiated by the addition of dimethyl sulphoxide (0·1 ml) to 0·1 ml in each well. The amount of yellow MTT that was reduced to purple formazan was measured spectrophotometrically at an absorbance of 550 nm. This reduction takes place only when mitochondrial reductase enzymes are active and relates directly to the number of viable cells.

Annexin/propidium iodide (PI) staining. After 1 day of culture with IL-15 and/or IL-21, IELs were stained with annexin–fluorescein isothiocyanate (FITC) conjugate (3 μl in 100 μl of binding buffer; Invitrogen, Carlsbad, CA) and PI (50 μg/ml) and then analyzed by flow cytometry (Beckman Coulter FC500; Beckman Coulter, Miami, FL).

Carboxyfluorescein succinimidyl ester (CFSE) staining. CFSE-loaded IELs (10 μm; Invitrogen) were cultured for 7 days with the cytokine stimuli before being stained with PI and analyzed by flow cytometry.

Proliferation. Proliferation after incubation for 3 days with IL-15 and/or IL-21 was measured by the incorporation of tritiated thymidine ([3H]TdR) after a 6-hr pulse, as described previously.5,32

Cytotoxic activity. To measure IEL cytotoxic activity after 3 days of culture with IL-15 and/or IL-21, target cells (P815 cells, Jurkat cells and WEHI cells, all from the American Type Culture Collection, Rockville, MD) were labelled with [51Cr]sodium chromate (New England Nuclear, Boston, MA) and then cultured with IELs for 4 hr at an effector:target cell ratio of 50:1. IELs were supplemented with anti-CD3 IgG immunoglobulin (50 ng/ml; R&D Systems) when directed against the P815 cells. The percentage of cytotoxicity was calculated from the specific release of [51Cr]sodium chromate relative to the spontaneous and maximal release of [51Cr]sodium chromate with medium and cetrimide, respectively, as described previously.6

Serine esterase release. Serine esterases were detected using the N-α-benzyloxycarboxyl-l-lysinethiobensyl ester (BLT; Calbiochem, San Diego, CA) assay, as described previously.8 In brief, after a 3-day stimulation with cytokines, IELs were cultured for 4 hr with anti-CD3 IgG immunoglobulin (50 ng/ml) in medium lacking phenol red and the supernates were collected. To each sample was added 0·2 mm 5,5′-dithio-bis(2-nitrobenzoic acid) and 0·2 mm BLT in phosphate-buffered saline. During a 45-min incubation in the dark, granzymes in each sample cleave BLT, causing a yellow color that can be quantified by absorbance measurements at 410 nm after the subtraction of background.

Immunofluorescence detecting surface markers. IELs, cultured for 3 days with the cytokine stimuli, were stained by immunofluorescence for CD2, CD3, IL-2 receptor alpha (CD25), CD27, CD28, CD44, CD62L, CD94, CCR7, NKG2D, TCR-αβ, or an isotype-matched immunoglobulin G (IgG) control (all from R&D Systems). This was followed by incubation with goat anti-mouse IgG conjugated to FITC and the immunofluorescence was read by flow cytometry. The relative fluorescence intensity (RFI) is the fold increase in staining compared with an IgG–FITC control.

Enzyme-linked immunosorbent assay (ELISA). After the IELs were cultured for 3 days with IL-15 and/or IL-21, the supernates were tested for IFN-γ, FL and TNF-α production by ELISA (R&D Systems).

Detection of STAT and mitogen-activated protein (MAP) kinase phosphorylation. At various time-points, cells were permeabilized (Cytofix Cytoperm; Pharmagen, San Diego, CA), then stained with antibodies against phosphorylated STAT1, STAT3, STAT4 and STAT5 (R&D Systems) followed by rabbit anti-goat IgG conjugated to FITC. Alternatively, they were stained for p38 or extracellular signal-regulated kinase (ERK) (Invitrogen) followed by goat anti-rabbit IgG conjugated to FITC. The fluorescence was read by flow cytometry.

In other experiments, nuclear extracts were isolated using a Nuclear Extract Kit (Active Motif, Carlsbad, CA) and samples were separated by electrophoresis on a 10% sodium dodecyl sulfate polyacrylamide gel, transferred to nitrocellulose paper and probed antibodies against phosphorylated STAT or MAP kinase proteins.

Statistics

Groups of data were compared using analysis of variance (anova) with the Tukey post-test to compare pairs of data sets within the group. The data are expressed as the mean value and standard error of the mean.

Results

IL-21 has no effect on granularity, survival, or proliferation of IELs

IL-21 has been shown to increase the size and granularity of NK and NK T cells.13,22,33 Therefore, the plot displaying these characteristics by forward scatter and side scatter, respectively, was compared after culture with the cytokine stimuli (Fig. 1). This plot showed that the IELs are highly purified and found in the same region of the scatterogram as PBLs. The scatterogram for IELs was the same regardless of the cytokine stimulus, indicating no change in the size and granularity of the IELs.

Figure 1
A typical plot displaying forward scatter (FS) versus side scatter (SS) is demonstrated for intra-epithelial lymphocytes (IELs) and peripheral blood lymphocytes (PBLs) cultured for 3 days with medium alone or with the cytokine stimuli (10 ng/ml) as noted. ...

IL-21 promotes the apoptosis of NK and T cells, but not of NK T cells or CD8+ T cells.13,16,18,21,22 Therefore, its effects on the numbers of viable IELs and on apoptosis were determined. According to the MTT assay, which measures the number of viable lymphocytes, IL-15 increases, whereas IL-21 does not change, this parameter (Fig. 2). Annexin/PI staining was then performed to determine whether IL-15 and/or IL-21 have prosurvival effects on IELs 34 (Fig. 3). There were fewer apoptotic and necrotic cells following stimulation with IL-15 than with medium alone (9 ± 2% versus 26 ± 4%, P < 0·05, n = 3). Following stimulation with IL-21, the number of apoptotic and necrotic cells (22 ± 3%) was no different from medium alone. The combined stimulus of IL-15 and IL-21 induced fewer numbers of annexin+ and PI+ cells (10 ± 2%) than did IL-21 alone (P < 0·05).

Figure 3
Intra-epithelial lymphocytes (IELs) were cultured for 3 days with interleukin (IL)-15 and/or IL-21 (10 ng/ml), then stained with annexin–fluorescein isothiocyanate (FITC) conjugate and propidium iodide (PI). The numbers represent the percentage ...
Figure 2
Intra-epithelial lymphocytes (IELs) were cultured for 3 days with interleukin (IL)-15 and/or IL-21 (10 ng/ml), then tested for the number of viable cells using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (expressed as ...

Cell division was then analyzed by CFSE staining on day 7 (Fig. 4). Stimulation with IL-15 slightly increased the numbers of IELs in cell division. Stimulation with IL-21 had no effect alone, but increased when combined with IL-15.34

Figure 4
Carboxyfluorescein succinimidyl ester (CFSE)-loaded intra-epithelial lymphocytes (IELs) were cultured with interleukin (IL)-15 and/or IL-21 (10 ng/ml) for 7 days then stained with propidium iodide (PI) and analyzed by flow cytometry. The histograms represent ...

IL-21 increases the proliferation of naïve but not of memory T cells 12 and counteracts the expansion of NK cells and CD44hi CD8+ T cells.21,22 To evaluate this for IELs, proliferation was determined by measurement of [3H]TdR incorporation (Fig. 5). On day 3, proliferation increased to an equal extent with three concentrations of IL-15 (1, 10 and 100 ng/ml) compared with medium alone.5 IL-21 did not alter proliferation whether tested alone or with IL-15 at the same concentrations.

Figure 5
Intra-epithelial lymphocytes (IELs), cultured for 3 days with interleukin (IL)-15 and/or IL-21 (10 ng/ml), were pulsed for 6 hr with tritiated thymidine and incorporation of radiolabel was measured. The mean and standard error of the mean are shown for ...

IL-21 has been shown to inhibit IL-15-induced proliferation independently of the CD3–TCR ligation but to enhance CD3–TCR-induced proliferation.21 Therefore, IELs were stimulated with anti-CD3 supplemented with IL-15 and/or IL-21 (Fig. 5). Proliferation increased with CD3 ligation and even more in the presence of IL-15. Again, it was not affected by IL-21, either alone or with IL-15. Of note, the amount of cell division was greater with IL-15 than with anti-CD3 IgG immunoglobulin alone, as shown previously,32 indicating the propensity of IELs to respond to a greater extent to the cytokine milieu rather than to cognate antigen.

IL-21 up-regulates perforin-mediated cytotoxic activity

IELs, cultured for 3 days with three concentrations of IL-15 and/or IL-21 (1, 10, or 100 ng/ml), were tested for CD3-triggered cytotoxic activity against P815 cells (Fig. 6a illustrates results with 10 ng/ml). There was a modest increase in redirected lysis with IL-15 but a marked augmentation with IL-21 at all three concentrations tested.

Figure 6
(a) Intra-epithelial lymphocytes (IELs), cultured for 3 days with interleukin (IL)-15 and/or IL-21 (10 ng/ml), were incubated for 4 hr with radiolabelled P815 cells (in the presence of 50 ng/ml of anti-CD3 IgG immunoglobulin) at an effector:target cell ...

To measure serine esterase release, IELs, after 3 days of culture with IL-15 and/or IL-21, were exposed to anti-CD3 for 4 hr and the supernates tested using the BLT assay (Fig. 6b illustrates results with 10 ng/ml). Again, IL-15 (at 10 and 100 ng/ml) increased serine esterase release, but IL-21 (at all three concentrations) had a much greater effect.

Perforin-mediated lysis and serine esterase release are mediated through the CD3–TCR complex. Therefore, expression of this complex was measured after stimulation with cytokine (Fig. 6c). Fresh IELs were 99 ± 5% CD2+, 88 ± 8% CD3+, 88 ± 8% TCR-αβ+ and 85 ± 5% CD8+ with only 4 ± 2% CD4+ T cells. Culture with IL-15 and/or IL-21 did not affect the expression of these markers (data not shown).

IL-21 has no effect on cytotoxicity mediated through FL or TNF-α

IELs, stimulated for 3 days with IL-15 and/or IL-21, were exposed to radiolabelled Jurkat cells, a target specifically susceptible to FL-mediated lysis (Fig. 7a). IL-15, when combined with IL-21, increased lysis compared with IL-21 alone. Similarly, the concentration of FL, measured using ELISA, increased with IL-15 in the culture (Fig. 7b). IL-21 alone had no effect.

Figure 7
(a) Intra-epithelial lymphocytes (IELs), cultured for 3 days with interleukin (IL)-15 and/or IL-21 (10 ng/ml), were incubated for 4 hr with radiolabelled Jurkat cells at an effector:target cell ratio of 50:1. Cytotoxicity was determined by the release ...

IELs, stimulated for 3 days with IL-15 and/or IL-21, were exposed to radiolabelled WEHI cells, a target specifically susceptible to TNF-α-mediated lysis (Fig. 8a). The cytokine stimuli did not alter this low-level cytotoxic activity, although these cells are sensitive to TNF-α-mediated cytotoxicity (10 ng/ml of TNF-α induces 23 ± 12% cytotoxicity at 4 hr). Only minimal extracellular TNF-α could be detected with IL-15 and/or IL-21 (Fig. 8b).

Figure 8
(a) Intra-epithelial lymphocytes (IELs), cultured for 3 days with interleukin (IL)-15 and/or IL-21 (10 ng/ml), were incubated for 4 hr with radiolabelled WEHI cells at an effector:target cell ratio of 50:1. Cytotoxicity was determined by the release of ...

IL-21 decreases IL-15-induced expression of CD25, CD44 and CD94, but does not alter the expression of CD28, NKG2D or IFN-γ

To determine whether T-cell activation was affected by IL-21, IELs were stained for CD25 after 3 days of culture with IL-15 and/or IL-21 (Fig. 9). There was a slight increase in CD25 following culture with IL-15, which was reduced by incubation with IL-21. Similarly, CD44 and CD94 expression increased with IL-15 and decreased with the combination of IL-15 and IL-21. This contrasts with the literature showing that IL-21 increases CD94 expression in NK and NKT cells.13,22,29

Figure 9
Intra-epithelial lymphocytes (IELs), cultured with interleukin (IL)-15 and/or IL-21 (10 ng/ml), were stained by immunofluorescence for surface markers after 3 days. The numbers in the graphs represent the relative fluorescence intensity (and are representative ...

IELs are effector memory T cells that are CD27 CD28 CD44high 62Llow CCR7. These surface markers were not altered by culture with IL-15 and/or IL-21 except for CD44 as stated above (Fig. 9). The numbers of CD28+ IELs ranged from 0 to 4% and were not affected by IL-15 or IL-21 (data not shown) despite up-regulation of this marker by IL-21 in other systems.20,33,35

NKG2D expression increased as a result of stimulation with IL-15 but was unchanged following stimulation with IL-21, although the degree of expression was low (RFI ranging from 1·8 to 2·0). This contrasts with the reports showing that IL-21 reduces its display on human NK and CD8+ T cells.33

IFN-γ production increased upon stimulation with IL-15 but was unaffected by IL-21 (Fig. 10). Supplementing with anti-CD3 IgG immunoglobulin did not alter the effects of IL-15 and IL-21 on IFN-γ production (data not shown).

Figure 10
Intra-epithelial lymphocytes (IELs) were cultured with interleukin (IL)-15 and/or IL-21 (10 ng/ml) for 3 days. The culture supernates were collected and tested for interferon-γ (IFN-γ) by enzyme-linked immunosorbent assay (ELISA) (mean ...

STAT proteins, ERK and p38 are minimally phosphorylated by IL-15 and IL-21

STAT3 and STAT5 phosphorylation characteristically occurs with γc cytokines. However, when IELs were cultured with IL-15 and/or IL-21 for 0·5, 1, 2, 4 or 18 hr, STAT1, STAT3, STAT4, STAT5, ERK and p38 were minimally phosphorylated, except for a 1·4-and 2-fold increase in fluorescence intensity for STAT5 with IL-15 at 4 hr (Fig. 11). p38 was minimally phosphorylated, while ERK was negative although this could be a result of the low sensitivity of the method. Western blot analysis revealed no phosphorylation because it is probably not as sensitive as flow cytometry (data not shown).

Figure 11
Intra-epithelial lymphocytes (IELs), cultured with interleukin (IL)-15 and/or IL-21 (10 ng/ml), were stained by immunofluorescence for intracellular markers after 4 hr. The numbers in the graphs represent the relative fluorescence intensity (and are representative ...

Discussion

Human IELs represent a unique compartment of lymphocytes that differ from murine IELs and from other human lymphocytes. This study showed that IL-21 has variable effects depending upon the function studied. It increases perforin-mediated redirected lysis and serine esterase release, suggesting that this may play a role in tumour immunology. In contrast, IL-21 had no effect on FL-or TNF-α-mediated lysis. It can, in fact, decrease IL-15-induced expression of CD25, CD44 and CD94. These changes occurred despite the increase in proliferation and survival induced by IL-15 but not by IL-21.

IL-21 up-regulates perforin-mediated lysis in most other systems 13,19,21,24,26,28,29 except for one.31 Similarly, perforin-mediated lysis and serine esterase release by IELs are promoted by IL-21. In contrast, IL-21 had no effect on FL-or TNF-α-induced cytotoxicity by IELs; the effects of IL-21 on these actions have not been studied using any other cell type. IL-15-induced lysis of colonic adenocarcinoma cells by IELs is mediated by perforin and FL.8 The up-regulation of perforin-mediated events by IL-21 may make IELs effective at tumour lysis. IL-21 has been shown to prolong tumour-free survival of animals injected intraperitoneally with thymoma and reduces the tumour burden in mice injected in the flank with colon cancer cells or intravenously/intrasplenically with melanoma, suggesting that this cytokine may serve as an effective anti-tumour agent.18,22,36 Tumour control in these models is perforin-mediated by CD8+ T cells or NK cells.

IL-21 enhances both the proliferation of T cells costimulated with anti-CD3 and the antigen-dependent proliferation of human cytomegalovirus (CMV)-specific CD8+ T cells.16,21,37 In addition, it promotes the growth of effector memory (CD44high 62Llow) CD8+ T cells, the same phenotype as IELs. However, IL-21 had no effect on the apoptosis, necrosis, proliferation, or cell division of IELs, regardless of whether or not IL-15 was present. It did not change the prosurvival effects of IL-15. Therefore, it does not expand or control the IL-15-induced expansion of CD3-triggered IELs, unlike its role in other systems.19,21

Some have suggested that IL-21 down-regulates the innate immune response while enhancing the adaptive immune response. It may serve as a third signal with antigen and costimulation.27 However, IL-21 had no effect on the proliferation or IFN-γ production by CD3-activated IELs. Probably, the effects of this cytokine differ depending upon the state of differentiation of the lymphocytes.

IFN-γ generation was up-regulated by IL-15, as shown previously 5 and as typical of memory T cells. IL-21 enhances both cytotoxic activity and IFN-γ production by NK and CD8+ T cells.16,19,21,26 Similarly, there is a defect in IFN-γ+ cells in IL-21R−/− mice.19 In contrast, IL-21 caused naïve CD8+ T cells to differentiate into cytotoxic effector T cells with little ability to synthesize IFN-γ.27 The IL-21-mediated regression of tumours was inhibited or, in contrast, not affected by an IFN-γ-deficient environment.25,26 IELs represent a third pattern with IL-21 stimulating potent cytotoxicity but not affecting their easily measurable IFN-γ production. If tumour rejection occurs in an IFN-γ-independent manner, it could have important clinical implications because the production of IFN-γ during overwhelming anti-tumour responses is accompanied by severe side effects.38

The effects of IL-21 on IELs are not mediated through STAT1, 3, 4, or 5. Moreover, the MAP kinase ERK is not affected, as shown in a promyelocytic cell line.39

IL-21 mimics some of the effects of IL-15, namely the promotion of perforin-mediated killing and serine esterase release. However, it lacks the ability of IL-15 to up-regulate IFN-γ production and to promote survival. IL-15 may drive lymphocytes to become long-lived memory cells, whereas IL-21 may steer them into an effector cell pathway.

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