Granulysin is a cytolytic effector molecule used by lymphocytes to kill tumor and microbial cells. Regulation of granulysin production is complex. A significant delay (5 days) following stimulation of CD4+ T cells with IL-2 occurs before granulysin is produced. Unfortunately, the mechanisms responsible for this delay are unknown. We have recently demonstrated that granulysin-mediated killing of Cryptococcus neoformans by CD4+ T cells is defective during HIV infection. This is because CD4+ T cells from HIV-infected patients fail to produce granulysin in response to IL-2 activation. The present studies examined the mechanism of delayed production of granulysin and the mechanism of the defect in HIV patients. We demonstrate that IL-2 initially requires both STAT5 and PI3K activation to increase expression of IL-2Rβ, produce granulysin, and kill C. neoformans. The increased expression of IL-2Rβ precedes granulysin, and preventing the increased expression of IL-2Rβ using small interfering RNA knockdown abrogates granulysin expression. Moreover, following the increased expression of IL-2Rβ, blocking subsequent signaling by IL-2 using IL-2Rβ-specific blocking Abs abrogates expression of granulysin. Finally, CD4+ T cells from HIV-infected patients, who are defective in both STAT5 and PI3K signaling, fail to express IL-2Rβ and fail to produce granulysin. These results suggest that IL-2 signals via PI3K and STAT5 to increase expression of IL-2Rβ, which in turn is required for production of granulysin. These results provide a mechanism to explain the “late” production of granulysin during normal T cell responses, as well as for defective granulysin production by CD4+ T cells in HIV-infected patients.
Granulysin is a member of the saposin-like protein family of lipid binding proteins. Along with perforin and granzymes, it is a constituent of the cytolytic granules of human CTL and NK cells (1). Granulysin can disrupt artificial liposomes and cell membranes, damage mitochondria, and activate caspase-9 to induce apoptosis (2). Additionally, granulysin exhibits potent cytotoxic activity against tumor cells and a broad panel of microbial pathogens including bacteria, fungi, and parasites (1, 3, 4). Moreover, our previous data have demonstrated that granulysin is the effector molecule used by IL-2-activated CD4+ T cells to kill Cryptococcus neoformans (5), which is one of the most important microbial pathogens in patients with compromised CD4+ T cell-dependent immunity, such as AIDS (6). It follows that CD4+ T cells from HIV-infected patients have defective activation and do not kill C. neoformans (5). Accordingly, granulysin appears to play a substantial role against tumor growth and microbial infections.
Granulysin is constitutively expressed in primary NK cells (7) and IL-2-dependent, Ag-driven functional T cell lines (8); however, stimulation with IL-2 does not enhance the granulysin expression in NK cells (7). In contrast, resting human PBL show a very low level of granulysin expression, but granulysin expression is remarkably induced in PBL stimulated by antigenic or mitogenic stimulation (8). However, the precise regulatory mechanism underlying granulysin expression in T cells or NK cells has not been well addressed. In this study, we examine the mechanism that drives CD4+ T cells to express granulysin and hope to identify potential targets for therapeutic interventions to restore the ability of CD4+ T cells in HIV-infected patients to express granulysin and provide anticryptococcal activity.
IL-2, a growth factor for Ag-stimulated T lymphocytes, has pleiotropic actions on the immune system and plays a vital role in the modulation of immune responses. The IL-2 receptor (IL-2R) is composed of α, β, and the common cytokine receptor γ (γc) subunits (9). IL-2Rβ (CD122) and IL-2Rγ (CD132) subunits combine to form the intermediary affinity IL-2R, whereas the addition of the IL-2Rα-chain results in the formation of a high-affinity IL-2R (9). Expression of the gene encoding IL-2Rα is undetectable in resting T cells but is strongly induced with T cell activation (10). The IL-2Rβ gene is expressed constitutively in NK cells and CD8+ T cells, but it is expressed at very low levels on CD4+ T cells. Upon activation of CD4+ T cells, IL-2Rβ is induced, which presumably plays a regulatory role (11, 12, 13). By contrast, IL-2Rγ is expressed constitutively on T cells (14, 15). The cytoplasmic domains of the IL-2R subunits do not possess intrinsic enzymatic activity. Thus, the actions of IL-2 are mediated by signal transduction cascades that are initiated by IL-2-induced oligomerization of IL-2Rα, IL-2Rβ, and IL-2Rγ subunits on activated T cells (16, 17). This juxtaposes cytoplasmic Janus family tyrosine kinases Jak1 and Jak3 that associate with IL-2Rβ and IL-2Rγc, respectively (16, 17, 18). Activated Jak kinases phosphorylate specific tyrosine residues in IL-2Rβ that serve as docking sites for SH2 domain-containing signaling molecules, such as STAT5a, STAT5b, and Shc (17). In addition to this conventional pathway, activation of other signaling pathways, including PI3K- and Ras-MAPK-coupled pathways, leads to the transcription of target genes that contribute to IL-2-dependent biologic actions (16, 17).
In the present study, we investigated the mechanism of granulysin expression in IL-2-stimulated CD4+ T cells. The role of signaling pathways was examined using immunoblot with and without pharmacologic inhibitors. Expression of IL-2R subunits was examined using RT-PCR, immunoblot, and flow cytometric techniques. The contribution of IL-2R subunits was assessed using blocking Abs and gene knockdown by small interfering RNA (siRNA).3 The impact of defective signaling on microbicidal activity was assessed by examining cryptococcal killing (5). Finally, CD4+ T cells from HIV-infected patients were used to assess the mechanism of the defect in these patients.
Materials and Methods
Goat anti-IL-2Rα, anti-IL-2Rβ, anti-IL-2Rγ Abs and mouse anti-IL-2Rβ-PE mAbs were purchased from R&D Systems. Anti-phospho-STAT5 (Tyr694) (14H2), anti-STAT5, anti-phospho-Akt (Ser473) (587F11), anti-Akt (rabbit polyclonal antibodies), anti-phospho-p38 MAPK (Thr180/Tyr182) (3D7), anti-p38 MAPK (L53F8), anti-phospho-SAPK/JNK (Thr183/Tyr185) (G9), and anti-SAPK/JNK (56G8) mAbs were all obtained from Cell Signaling Technology. Secondary Abs depend on the detection methods (see below).
The following inhibitors used: 10 μM PI3K inhibitor LY294002; 1 μM JAK3 inhibitor VI, which prevents IL-2-induced phosphorylation of JAK3 and STAT5; 20 μM MAPK/ERK kinase 1 (MEK1) inhibitor PD98059; 1 μM JNK inhibitor II SP600125; and 10 μM p38 kinase inhibitor SB203580 (Calbiochem). Concentrated stocks of 100 mM inhibitors were prepared in DMSO or H2O and stored at −20°C. Two hours before IL-2 stimulation, the CD4+ T cells were incubated at 37°C with pharmacological inhibitors. In all cases, untreated cells behaved similarly to those treated with DMSO alone.
CD4+ T cell isolation and activation
Human heparinized blood samples were collected by venipuncture from healthy young adults or antiretroviral therapy-naive HIV-infected young adults. These patients were selected from a cohort of HIV-infected individuals with CD4+ T cells counts from 400 to 1000/μl, so that enough cells could be recovered to perform experiments. No other criteria were used to exclude participants. These HIV patients had no other comorbidities; none of these patients had documented cryptococcal infection and the viral load was low (undetectable, 75 × 103/mm3). PBMC were prepared immediately after collection by separating over a Ficoll-Hypaque density gradient (Sigma-Aldrich) as described previously (19). PBMC were harvested and washed three times in HBSS (Invitrogen) followed by subset isolation.
CD4+ T cells were isolated with the CD4+ negative isolation kit (Miltenyi Biotec) using the autoMACS separator (Miltenyi Biotec) following the manufacturer's instructions. The purified CD4+ T cells were resuspended in medium containing RPMI 1640 (Invitrogen), 5% human AB serum (BioWhittaker), 100 U/ml of penicillin, 100 μg/ml of streptomycin, 0.25 μg/ml of amphotericin B, 2 mM l-glutamine, 1 mM sodium pyruvate, and 0.1 mM nonessential amino acids (all from Invitrogen). The purity of CD4+ T cells from healthy donors and HIV-infected patients was routinely >95 and 90%, respectively, and the contamination of CD8+ T cells or NK cells was routinely <1% as determined by flow cytometry with Guava EasyCyte (Guava Technologies). All of the study subjects participated voluntarily and gave informed consent, and human research was conducted in accordance with the approval of the conjoint Board of Medical Ethics at the University of Calgary.
CD4+ T cells were stimulated with IL-2 at concentration of 100 U/ml (R&D Systems). The viability of cells was not altered by all treatments as assessed by trypan blue exclusion and Guava ViaCount Reagent (Guava Technologies).
Western blot analysis
Following stimulation, cold washing medium (PBS, 1% NaF, 0.5% Na3VO4, 10% sodium pyrophosphate) was added to terminate stimulation and cells were centrifuged to generate a cell pellet that was lysed with lysis buffer (50 mM Tris (pH 6.8), 1% SDS, 0.025% bromophenol blue, 10% glycerol, 20 mM DTT) supplemented with a mixture of protease inhibitors (Calbiochem) and a mixture of phosphatase inhibitors (Calbiochem). Proteins from the lysates of 1 × 105 cells were loaded in each lane of a 4–12% gradient precast Tris-glycine gel (Invitrogen), separated by electrophoresis, transferred to a nitrocellulose membrane (Bio-Rad), and blotted with respective primary Abs. Polyclonal anti-granulysin Ab 519/GST rabbit serum has been previously described (5), and a mouse anti-human β-actin mAb was purchased from Chemicon International. The reactive bands were visualized with an Odyssey infrared imaging system (LI-COR Biosciences) using IRDye 800CW purified goat anti-mouse IgG (1/20,000 dilution) or with IRDye 700DX purified goat anti-rabbit IgG (1/10,000 dilution) (Rockland Immunochemicals).
IL-2R flow cytometry assay
FITC-conjugated AffiniPure F(ab′)2 fragment donkey anti-goat IgG (H+L) was purchased from Jackson ImmunoResearch Laboratories. CD4+ T cells were labeled with anti-IL-2Rα or IL-2Rγ Abs followed by FITC-anti-goat IgG (H+L) (donkey) or anti-IL-2Rβ-PE mAbs and analyzed by flow cytometry using the Guava EasyCyte flow cytometer. At least 5000 events were recorded during each analysis.
siRNA preparation and gene silencing assays
The siRNA sequences (an ON-TARGETplus SMARTpool with four separate siRNAs, catalogue no. L-007984–00-0005) targeting human IL-2Rβ (GenBank accession number NM_000878) were synthesized by Dharmacon. The sequences of CD20 and a nonsilencing siRNA, which served as negative controls, are 5′-AACCACTCTTCAGGAGGATGT-3′ (Dharmacon) and 5′-AATTCTCCGAACGTGTCACGT-3′ (Dharmacon), respectively. It has been shown that CD20 siRNA could decrease the constitutive expression of CD20 on Ramos B cells (a B cell line that constitutively expresses CD20) by ∼50% (Dr. J. P. Deans, unpublished observations). Transfection and siRNA-mediated knockdown conditions for CD4+ T cells were optimized as described previously (5). Briefly, CD4+ T cells were mixed with each individual siRNA (5 nM) and electroporated in a 0.4-cm cuvette using the Gene Pulser apparatus (Bio-Rad) with a pulse of 200 V and capacitance of 800 μF. The treated cells were stimulated with IL-2 as described above.
Total RNA was extracted from individual cell samples using an RNA extraction kit (Qiagen). The extracted total RNA was quantified by Thermo Spectronic (Thermo Scientific). One microgram of total RNA from each sample was reverse transcribed using the ImProm-II reverse transcription system (Promega) following the manufacturer's instructions. The following IL-2Rβ (NM_000878) or GAPDH-specific primers were used: 5′-TGGCCTTCAGGACAAAGCCT-3′ and 5′-TCCTCT GAGTAGGGGTCGTA-3′ (20); 5′-TCACCATCTTCCAGGAGCGA-3′ and 5′-AGTGATGGCATGGACTGTGG-3′. The PCR profile for IL-2Rβ and GAPDH was as follows: denaturing at 94°C for 1 min, followed by 25 cycles of denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. A final extension at 72°C for 5 min was used. The number of cycles was adjusted so that amplification occurred over the linear range. The PCR products were separated by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining.
C. neoformans CAP67 (ATCC 52817) was obtained from the American Type Culture Collection. The organisms were maintained on Sabouraud dextrose slants (Difco) and passaged to fresh slants every month as previously described (21). CFU were determined as previously described (22, 23, 24). C. neoformans (2 × 103/well (200 μl)) was incubated with or without 5 × 105 lymphocytes (E:T ratio of 250:1 unless otherwise specified). The number of CFU of C. neoformans per well was determined at 0, 24, or 48 h by lysing the effector cells with dH2O followed by diluting and spreading onto Sabouraud dextrose agar plates. Preliminary experiments established that dH2O lysed effector cells without affecting fungal viability. Results are expressed as CFU per milliliter. A decrease in the CFU of C. neoformans compared with the growth of C. neoformans alone indicates growth inhibition. Values lower than the inoculum indicate killing.
Data were expressed as means ± SEM. Each experiment was performed with different donors on different days. Statistical analysis was performed by using the ANOVA. For this purpose, the Fisher least-squares difference was used when allowed by the F test. Student's t test was used to do pairwise comparisions using the Bonferroni correction. For these tests, a p value of <0.05 was considered significant.
Characterization of the IL-2 signaling pathway in human peripheral blood primary CD4+ T cells
CD4+ T cells from HIV-infected patients fail to produce granulysin and kill C. neoformans following treatment with IL-2 (5). To explore the mechanism of this defect, the signaling pathway in response to IL-2 that mediated granulysin production was examined in healthy adults. Experiments initially examined the role of STAT5, Akt, ERK-1/2, p38, and p54JNK pathways in primary human CD4+ T cells. CD4+ T cells were treated with IL-2, and STAT5, Akt, ERK-1/2, p38, and p54JNK phosphorylation were analyzed by Western blot using specific Abs. Within 5 min of stimulation, CD4+ T cells displayed phosphorylation of STAT5 on residue Tyr695 and this phosphorylation declined after 15, 30, and 60 min (Fig. 1,A). Following this initial event, there was a second, sustaining phosphorylation of STAT5 on days 3, 4, and 5 during continuous IL-2 stimulation (Fig. 1 B). This suggested that a regulatory event occurs in response to the initial signal that results in a sustained amplification of STAT5 activation.
In addition to Jak3/STAT5 signaling, IL-2 stimulation also triggered phosphorylation of ERK-1/2, p38, and p54JNK following both short-term and long-term stimulation (Fig. 1, A and B). However, we were unable to detect the phosphorylation of Akt, as an indication of PI3K activation, in either short- or long-term stimulation. To pursue activation of PI3K in CD4+ T cells, the CD4+ T cells were stimulated with IL-2 for 1, 2, 3, 4, or 5 days. IL-2 was withdrawn for 24 h and CD4+ T cells were then restimulated with fresh IL-2 for 5 min. Akt could only be detected after 3 days of initial stimulation, and it peaked 4 days after initial stimulation (Fig. 1 C). These results suggested that IL-2 can transiently activate PI3K in primary CD4+ T cells after 3 days of prior stimulation.
Inhibition of either PI3K or STAT5 abrogates granulysin expression in IL-2-stimulated CD4+ T cells
Experiments were performed to determine which signaling pathway is involved in granulysin expression in CD4+ T cells. For this purpose, CD4+ T cells were stimulated with IL-2, and pharmacological inhibitors were applied to block the respective signaling pathways. Pharmacological inhibition of PI3K by LY294002, which competes with ATP for binding to the PI3K catalytic domain of p110 (25) or STAT5 by JAK3 inhibitor VI, which prevents IL-2-induced phosphorylation of JAK3 and STAT5 (26), abrogated the granulysin expression in IL-2-stimulated CD4+ T cells (Fig. 2,A). In contrast, pharmacological inhibition of ERK-1/2 by PD980095, which specifically inhibits ERK-1 and ERK-2 phosphorylation by blocking MEK activity (27); of p38 by SB203580, a highly specific, cell-permeable inhibitor of p38 kinase (28); or of p54 by JNK inhibitor II (SP600125), which has a >300-fold selectivity for isoform JNK over the related MAPKs, ERK-1, and p38-2 (29), did not affect the granulysin expression (Fig. 2,A). Consistent with previous reports (26, 30, 31), neither LY294002 nor the JAK3 inhibitor affected the total number of viable CD4+ T cells as assessed by trypan blue dye exclusion, even after 7 days of incubation (Fig. 2,B). Importantly, both PI3K and JAK3 inhibitors abrogated the granulysin-dependent killing of C. neoformans by IL-2-stimulated CD4+ T cells (Fig. 2 C) (5). These results demonstrate that PI3K and STAT5, but not ERK-1/2, p38, or JNK, are required for granulysin expression in IL-2-stimulated CD4+ T cells.
Increased expression of IL-2R α and β subunits correlates with granulysin expression
During these studies, we observed that granulysin expression was detected late (5–7 days) after IL-2 stimulation, which seemed to occur after an increase in the expression of IL-2Rβ. Therefore, the time course of expression of IL-2R subunits was examined by Western blot. The increased expression of IL-2Rα and IL-2Rβ was only detectable after 3 days of IL-2 stimulation and was slightly further enhanced at 5 days (Fig. 3,A). The expression of granulysin was detectable only after 5 days of IL-2 stimulation and was greater at 7 days (Fig. 3 B). These results demonstrated a sequential correlation between the increased expression of IL-2Rα and IL-2Rβ and granulysin.
Inhibition of either PI3K or STAT5 blocks the increased expression of IL-2R α and β subunits in IL-2-stimulated CD4+ T cells
Because most peripheral resting CD4+ T cells express very low levels of IL-2Rβ (11, 13), which are increased following stimulation with IL-2, experiments were performed to assess the affect of blocking PI3K or STAT5 on the expression of IL-2R subunits. Resting CD4+ T cells expressed low levels of IL-2Rα and IL-2Rβ, which increased after 3 days of stimulation with IL-2. In contrast, the expression of IL-2Rγ did not increase following IL-2 stimulation (Fig. 4,A) (14, 15). Pharmacological inhibition of PI3K by LY294002 or STAT5 by JAK3 inhibitor blocked the increased expression of both α and β subunits of IL-2R, whereas the γ subunit was constitutively expressed (14, 15). These results were confirmed by flow cytometry (Fig. 4 B). Thus, PI3K and STAT5 are required for the increased expression of IL-2Rα and IL-2Rβ in CD4+ T cells, which correlates with the signals required for granulysin expression in these cells.
Increased expression of IL-2Rβ is a prerequisite for granulysin expression
The previous experiment had indicated that IL-2 stimulation induced the increased expression of both α and β subunits of IL-2R and that this correlated with granulysin expression. To determine whether the increased expression of either IL-2Rα or IL-2Rβ was required for granulysin expression in IL-2-stimulated CD4+ T cells, two approaches were used. In the first approach, specific blocking Ab against IL-2R subunits was applied. Freshly isolated CD4+ T cells were incubated with anti-IL-2Rα, anti-IL-2Rβ, or anti-IL-2Rγ blocking Ab 2 h before IL-2 was added to the media. Blocking Ab for IL-2Rα (anti-CD25) did not affect the increased expression of IL-2Rα and IL-2Rβ, or granulysin 5 days later (Fig. 5,A). However, blocking Abs to IL-2Rβ or IL-2Rγ abrogated the increased expression of IL-2Rα, IL-2Rβ, and granulysin (Fig. 5 A). In contrast, an isotype-matched Ab had no affect on the expression of IL-2Rα, IL-2Rβ, or granulysin (data not shown). These results suggested that the increased expression of IL-2Rβ, but not IL-2α, is required for granulysin expression and that IL-2Rγ is also required.
In the second approach, the increased expression of IL-2Rβ was silenced by IL-2Rβ-specific siRNA. To improve the electroporation efficiency, the CD4+ T cells were stimulated with IL-2 for 1 day and subjected to electroporation with IL-2Rβ-specific siRNA. As a control, CD4+ T cells were transfected with CD20 siRNA, nonspecific siRNA, or sham transfection (−). After transfection, the CD4+ T cells were stimulated with IL-2 for another 4 days. siRNA abrogated the increased expression of IL-2Rβ at both the level of mRNA (Fig. 5,B) and protein (Fig. 5, C and D) but maintained the basal level of expression. As a consequence, granulysin expression was abrogated. By contrast, neither CD20 nor nonspecific siRNA affected the increased expression of IL-2Rβ or granulysin (Fig. 5, B–D). Two different IL-2Rβ siRNA were tested and provided similar results (data not shown), suggesting that this was not due to off-target effects.
Both PI3K and STAT5 signal pathways are required for granulysin expression after increased levels of IL-2Rβ
To determine whether IL-2R signaling is still required for granulysin expression after the increased expression of IL-2Rβ, CD4+ T cells were stimulated with IL-2 for 3 days, and the cells were then rested for 24 h in IL-2-free media. CD4+ T cells increased expression of IL-2Rα and IL-2Rβ after 3 days of IL-2 stimulation, but they did not express granulysin, nor did they express granulysin after 24 h rest (Fig. 6,A). The cells were then restimulated for another 48 h. After IL-2 restimulation for 48 h, CD4+ T cells expressed granulysin. By contrast, addition of blocking Ab to IL-2Rβ during the restimulation abrogated the granulysin expression. As a control, the isotype control Ab had no effect on the expression of granulysin (Fig. 6 A). Furthermore, the IL-2Rγ-blocking Ab also blocked the granulysin expression, but the IL-2Rα-blocking Ab did not (data not shown). These results demonstrate a requirement of IL-2Rβ and IL-2Rγ signaling for granulysin expression.
Additional experiments were performed to test whether PI3K and STAT5 signal pathways are necessary for the expression of granulysin in CD4+ T cells following increased expression of IL-2Rβ. CD4+ T cells were stimulated with IL-2 for 3 days to induce the increased expression of IL-2Rβ (Fig. 6,B). The pharmaceutical inhibitors specific for PI3K and JAK3 were then added for another 2 days in the presence of IL-2, and the granulysin expression was assessed. Pharmacologic inhibition of PI3K or Jak3/STAT5 abrogated the expression of granulysin (Fig. 6 B). The results suggested that both PI3K and STAT5 are not only indispensable for the increased expression of IL-2Rβ, but that they are obligatory for granulysin expression even after the increased expression of IL-2Rβ on CD4+ T cells with IL-2 stimulation.
Failed granulysin expression in IL-2-stimulated CD4+ T cells from HIV patients is due to defective PI3K and STAT5 pathways
In our previous work, we have shown that IL-2-stimulated CD4+ T cells from HIV-infected patients do not express granulysin and fail to kill C. neoformans (5); however, the defective mechanism was not clear. Experiments were performed to determine whether the defect in granulysin expression was due to defective STAT5 or PI3K signaling and defective expression of IL-2Rβ. Following short-term IL-2 stimulation, CD4+ T cells from HIV-infected patients demonstrated STAT5 phosphorylation (Fig. 7,A) that was similar to that of healthy adults (Fig. 1,A); however, during long-term stimulation, STAT5 phosphorylation was not seen (Fig. 7,B). Similar to healthy adults, we failed to detect Akt phosphorylation in short- or long-term stimulation (Fig. 7,B). However, Akt phosphorylation is also defective after restimulation with IL-2 (Fig. 7,C). Furthermore, IL-2 stimulation did not increase the expression of IL-2Rβ in HIV-infected patients compared with healthy adults (Fig. 7 D). Densitometry results also demonstrated that CD4+ T cells from healthy donors have significantly higher levels of expression of IL-2R than do those of HIV patients (data not shown). These results suggested that the defect in granulysin expression and cryptococcal killing is due to defective increased expression of IL-2Rβ and subsequent defective PI3K and STAT5 signaling.
In this study, we demonstrate that: 1) IL-2 activates STAT5, PI3K, ERK-1/2, p38, and JNK in human peripheral blood CD4+ T cells; 2) increased granulysin expression correlates with the increased expression of IL-2Rβ in primary CD4+ T cells; 3) both STAT5 and PI3K, but not p38, ERK-1/2, or JNK, are required for granulysin expression and increased expression of IL-2Rβ; 4) the increased expression of IL-2Rβ is a prerequisite for granulysin expression in IL-2-stimulated CD4+ T cells; and 5) IL-2-stimulated CD4+ T cells from HIV-infected patients have normal initial STAT5 phosphorylation but fail to increase expression of IL-2Rβ and are defective in phosphorylation of STAT5 and PI3K at later time points, and as a consequence have defective granulysin expression and killing of C. neoformans.
T cell lymphoma lines of murine and human origin are often used as versatile tools for immunologic research (32). Because the early exploratory work on STAT activation was done in transformed cell lines, sometimes with reconstructed receptors, the results may not reflect the specificity of activation in differentiated cells and tissues (33). Tumor cells may have altered signaling characteristics that may range from a constitutive basal activity within a “physiologic” context to complete alterations of individual pathways (34). For example, studies of the role of PI3K in T cell biology using leukemic cell lines might have misinterpreted the importance of this pathway for T cell signal transduction (35). Nevertheless, in cell lines, IL-2 stimulation activates the Ras/ERK, PI3K, and JAK/STAT cascades, and it also activates p38MAPK and p54MAPK/JNK (36). Thus, we thought it necessary to characterize the IL-2R signaling in human primary peripheral blood CD4+ T cells.
We demonstrated that IL-2R signaling activated STAT5, but not Akt, during short-term IL-2 stimulation. This can be explained by the observation that peripheral blood CD4+ T cells express very low levels of IL-2R α and β subunits (11, 13) and that PI3K signaling is too weak to be detected. During long-term IL-2 stimulation, Akt phosphorylation can only be detected transiently after 3 days of IL-2 incubation, correlating with the increased expression of IL-2R. Similarly, strong phosphorylation of STAT5 was only found after 3 days of IL-2 incubation and was sustained over periods of time, which is consistent with the time course of Akt activation (Fig. 1 C).
It is interesting to find that both LY294002 and Jak3 inhibitor VI abrogated granulysin expression. There are a number of mechanisms by which PI3K and STAT5 work cooperatively, which therefore might explain why both are required to induce granulysin expression. The PI3K pathway contributes to T cell proliferation mediated by the IL-2R pathway by potentiating, not triggering, mitogenic signaling by STAT5 (37). Additionally, STAT5 and PI3K pathways play distinct, coordinated roles in the IL-2R-mediated induction of cyclin D2, where PI3K is required for optimal transcription of STAT5-inducing cyclin D2 (38). Moreover, STAT5 in its phosphorylated form interacts with the p85 subunit of the PI3K, further suggesting that there is cooperation between STAT5 and PI3K (39).
Surprisingly, our understanding of IL-2Rβ regulation in primary T cells is relatively limited. Previous studies showed that IL-2Rβ was induced by IL-2 in B cells and that this promotes growth of B cells and small T cells (40, 41). Herein, we demonstrate that IL-2 stimulation via STAT5 strongly induced the expression of IL-2Rβ (Fig. 3,A) and CD4+ T cell growth (Fig. 2 B). STAT5 protein is a transcription factor that promotes IL-2-activated transcription of genes, such as IL-2Rα; however, the IL-2Rβ promoter does not have a STAT5 binding motif (42). Although the mechanism by which STAT5 contributes to IL-2Rβ induction is unknown, STAT5 does form a complex with the transcription factor Ets, which contains a binding site for the IL-2Rβ promoter, and the complex is important in IL-2 signaling (43).
The other identified pathway, PI3K, regulates diverse functions, including protein synthesis, glucose metabolism, and cell cycle control (44). Many Akt substrates are transcription factors or are directly involved in the regulation of gene transcription (45). PI3K is required for expression of Ets in T cells (46), and Ets and Sp-1 transcription factors in other cells (47, 48), which are important in IL-2Rβ gene regulation (49, 50).
Our previous studies have shown that granulysin is a late-expressed protein in CD4+ T cells after IL-2 stimulation (5). In this study, we demonstrated that the increased expression of IL-2Rβ occurred after 3 days of IL-2 incubation. Experiments were therefore performed to determine whether there was a critical step in induction of IL-2Rβ. We found that Abs specific for IL-2Rβ or IL-2Rγ blocked IL-2R signaling and granulysin expression, while in contrast Abs specific for IL-2Rα had no effect. We think that this is most consistent with a mechanism whereby IL-2Rα is not required. It is unlikely that saturating concentrations of IL-2 are responsible because previous studies have demonstrated that lower concentrations of IL-2 failed to induce granulysin expression in CD4+ T cells (51) and optimal killing of C. neoformans by IL-2-stimulated lymphocytes requires a minimum IL-2 concentration of 100 U/ml (52). When RNAi was applied to specifically inhibit the increased expression of IL-2Rβ, we were able to maintain the constitutive levels of IL-2Rβ but block the induced IL-2Rβ mRNA and protein expression. CD4+ T cells undergoing IL-2R signaling with basal levels of IL-2Rβ could not drive granulysin expression.
CD4+ T cells from HIV-infected patients are defective in granulysin expression after IL-2 stimulation (5). Most of the evidence in HIV infection indicates that constitutive expression of IL-2Rα/β/γ on CD4+ T cells is similar to healthy adults (53, 54, 55). The present studies show that CD4+ T cells from HIV-infected patients stimulated with IL-2 failed to induce the expression of IL-2Rβ and to activate both Akt and STAT5. This is consistent with a previous report showing that there is defective JAK/STAT5 signaling following Env ligation of CD4 (56). This was correlated with defective expression of granulysin. This finding supports a mechanism whereby basal levels of IL-2Rβ are not sufficient for granulysin expression in CD4+ T cells and that the increased expression of IL-2Rβ in CD4+ T cells is essential for granulysin expression.
During HIV infection, chronically activated CD4+ T cells are characterized by an anergic state with reduced proliferative capacity to recall Ags, mitogens, or cytokines such as IL-2 and IL-4 (57). It has been proposed that this suppression of IL-2 signaling events is mediated, in part, by the interaction of viral envelope glycoproteins with their cellular receptors, such as gp120 ligation with CD4, resulting in defective T cell activation (58). Indeed, JAK3 is the downstream target of the ligation between gp120 and CD4, and its activation is inhibited after the ligation (58, 59). However, we do not think that this mechanism underlies the defective responses (described herein) of CD4+ T cells obtained from HIV-infected patients. First, CD4 T cells from healthy donors maintained their ability to express granulysin in response to IL-2 stimulation when cultured in the presence of gp120 protein from three different strains of HIV-1 (data not shown), even at high concentrations of gp120 (5 μg/ml). Furthermore, the anticryptococcal activity of IL-2-stimulated CD4+ T cells was not affected when activated in the presence of HIV-1 gp120 (data not shown) at concentrations previously demonstrated to inhibit T cell responses (60). Alternatively, it has been demonstrated that HIV-infected individuals bear a constitutively activated C-terminally truncated form of STAT5 (STAT5Δ) (61, 62). Of particular importance to the data presented herein, these STAT5Δ isoforms show resistance to proteasome-mediated degradation and enhanced ability to bind to DNA (63), and they have been suggested to act as dominant negative regulators of STAT5-dependent gene transcription (64), including potentially IL-2-induced expression of granulysin in CD4+ T cells from HIV-infected individuals.
Finally, in our previous paper, we demonstrated that CD4+ T cells from HIV patients failed to induce granulysin expression and resulted in defective killing or growth inhibition of C. neoformans. We speculate that defective CD4+ T cell-mediated microbicidal activity is necessary but not sufficient for the acquisition of cryptococcosis because the microbicidal activity of CD4+ T cells occurred at a stage of disease (400–1000 CD4+ T cells/mm3) before the stage of HIV disease when patients are predisposed to cryptococcosis.
In summary, we investigated the mechanism involved in granulysin expression in human primary peripheral blood CD4+ T cells. We found that both PI3K and STAT5 signaling pathways are required for granulysin expression and IL-2Rβ induction, and that IL-2Rβ induction is a prerequisite for granulysin expression; however, both signaling pathways are defective in CD4+ T cells from HIV-infected patients. This results in a failure to induce IL-2Rβ, failed granulysin expression, and ultimately a failure in this mechanism of microbial host defense.
We thank Dr. J. P. Deans for the generous gift of CD20 siRNA, Brenda Beckthold for assistance in recruiting and enrolling HIV patients for this study, and Danuta Stack, Martina Timm-McCann, and Tineka Asma-Schollaardt for technical assistance. We also thank the participants and patients from the Southern Alberta Clinic for cooperation.
The authors have no financial conflicts of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by grants from Canadian Institute of Health Research (to C.H.M.), Canadian Foundation for AIDS Research (to C.H.M.), Jessie Bowden Lloyd Professorship in Immunology (to C.H.M.), and Canadian Institute of Health Research Training Program in Immunology (to C.F.Z.).
Abbreviation used in this paper: siRNA, small interfering RNA.