Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells

Abstract

Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

Figure 1. Different IL-2 Signal Strengths Regulate Perforin and Granzyme B Expression to Establish CTL Function

(A) Kinetics of perforin and granzyme B mRNA expression. Purified CD8⁺ T cells from naive B6 mice were primed with anti-CD3 and anti-CD28, removed from the TCR stimulus after 2 days, and recultured in 10 or 100 U/ml IL-2. Total RNA was analyzed by blotting at the indicated times. Results are representative of more than three independent experiments. See also Figure S1. (B) Perforin (left) and granzyme B (right) protein expression. Whole-cell lysates were analyzed by immunoblotting at the indicated times. Total STAT5 content was used as a control. Granzyme B expression was determined by intracellular staining and flow cytometry on day 6. (C) Intracellular cytokine expression after restimulation. CD8⁺ T cells were restimulated on days 4 and 6 with 10 nM PMA + 1 µM ionomycin for 6 hr. (D) Flow cytometry-based cytotoxicity assay for cytolytic activity. GP33-pulsed EL4 targets were coincubated with effector P14 CD8+ T cells that were differentiated as in (A). CTL activity was blocked by incubation with 5 mM EGTA (data not shown). (E) Accumulation of CD8⁺ T cells after stimulation as described in (A). The data are the mean and standard deviation summarized from at least five differentiations. (F) Cytotoxic activity after brief restimulation. P14 cells cultured until day 5 in low IL-2 were restimulated for 2 hr with PMA and Ionmycin, washed, resuspended in T cell media without IL-2, and cultured overnight before analyzing CTL activity. (G) Perforin expression in primed CD8⁺ T cells cultured in 10 U/ml IL-2 upon restimulation on day 6. Lysates from cells left unstimulated (U) or restimulated with PMA (P) and ionomycin (I) as in (C) for 6 hr were analyzed by RNA blotting (left) and immunoblotting (right).

Figure 2. Reciprocal Regulation of Perforin and IL-7Rα Reexpression by IL-2

(A) The regulation of IL-7Rα (CD127) re-expression after priming. Purified naive P14 CD8+ T cells were stained with antibodies against CD127 ex vivo (Naive), after priming for 2 days with anti-CD3 + anti-CD28 (day 2) and culture in 0.1, 1, 10 or 100 U/ml IL-2. The MFI of CD127 staining is shown in each panel. The analysis is representative of at least three separate differentiations. See also Figure S2. (B) Graphical summary of IL-7Rα expression from the representative experiment in (A). (C) Cell division history based on CFSE dilution after priming and culture in IL-2 as in (A). (D) IL-7Rα and perforin mRNA expression in naive and primed P14 CD8+ T cells. Total RNA was extracted at the indicated time points from cells in (A). (E) Flow cytometric analysis of IL-2Rα and IL-7Rα coexpression.

Figure 3. IL-2 Regulates Blimp-1, Bcl6, and Eomes Expression, and Eomes Activates the Perforin Gene

(A) Intracellular staining of IL-2. Stainings are of unstimulated cells (outlined histogram) or cells restimulated for 4 hr with 10 nM PMA + 1 µM ionomycin (shaded histograms). Results from (A)–(E) are representative of at least two independent experiments. (B) Blimp-1, Bcl6, and IL-2Rα mRNA expression kinetics were analyzed by RNA blotting. (C) T-bet and Eomes mRNA expression kinetics were analyzed by RNA blotting. (D) Eomes and perforin expression in response to IL-2 after day 4. Cultured CD8+ T cells were harvested and washed on day 4 and switched from low IL-2 to high IL-2 and vice versa (switch) or returned to their original IL-2 media (control) for an additional 2 days. Whole-cell lysates were generated and analyzed by immunoblotting on day 6. (E) Perforin and granzyme B (GrzmB) mRNA expression upon Eomes-VP16 transduction. On day 6 of culture in 10 U/ml IL-2, total RNA was analyzed by RNA blotting; uninfected (uninf), Eomes-VP16 (Eo-VP), or empty cassette (GFP) transduced; Th1 and NIH 3T3 cells are shown as controls. Transduction efficiency in each culture was equivalent between constructs. See also Figure S3.

Figure 4. IL-2 Regulates RNA Polymerase II Recruitment and the Binding of STAT5 and Eomes to Prf1

(A) ChIP analysis of endogenous STAT5. Chromatin was isolated from cells on day 6. The efficiency of recovery relative to input at the −1 kb region of Prf1 was 0.39% in high-IL-2 conditions for STAT5. The data in panels (A) and (B) show the mean and standard deviation of duplicate measurements, pooled from at least two immunoprecipitations of chromatin prepared from two independent CD8⁺ T cell differentiations. See also Figure S4. (B) ChIP analysis of endogenous Eomes. The efficiency of recovery relative to input for the −1 kb region of Prf1 was 0.97% in high-IL-2 conditions for Eomes. (C) ChIP analysis of RNA pol II. Data show the mean and standard deviation of duplicate measurements from separate immunoprecipitations from at least two independent differentiations; data from each differentiation was normalized based upon binding of RNA pol II to the Hprt TSS. The efficiency of recovery of the Hprt TSS was ~0.6% and was approximately six times greater than from the Ifng TSS. (D) ChIP analysis of RNA pol II at the Il7ra TSS from the chromatin analyzed in (C).

Figure 5. IL-2Rα-Deficient CD8⁺ T Cells Are Impaired in CTL Differentiation upon LCMV Infection

(A) Il2ra^+/+ and Il2ra^−/− mixed bone marrow chimeric (BMC) mice were infected with LCMV. Eight days postinfection, Il2ra^+/+ and Il2ra ^−/− CD44^hi CD8⁺ effector cells were sorted and their ability to kill GP33 peptide-pulsed target cells was assayed. (B) Quantitative RT-PCR analysis of cDNA prepared from sorted CD44^hi Il2ra^+l+ and Il2ra^−/− CD8⁺ T cells. Values were normalized based on amplification of Hprt mRNA. (C) Percent of total Il2ra^+/+ (upper number) and Il2ra^−/− (lower, gray number) CD8⁺ T cells from mixed BMC mice that express the indicated proteins was determined 6 days post-LCMV infection. CD25 expression by the Il2ra^+/+ cells is depicted. Dashed line shows staining in uninfected BMC mice. (D) Intracellular cytokine production of IL-2 and IFNγ by Il2ra^+/+ and Il2ra^−/− effector CD8⁺ T cells. Splenocytes from uninfected BMC mice and BMC mice on day 6 postinfection were stimulated for 4 hr with GP33 peptide, or PMA (50 ng/ml) and Ionomycin (500 ng/ml). Red numbers indicate the percentage of IL-2⁺ events among IFNγ⁺ cells. (E) Quantitative RT-PCR analysis of cDNA prepared from sorted CD44^hi Il2ra^+/+ and Il2ra^−/− CD8⁺ T cells. Values were normalized based on amplification of Hprt mRNA. Panels (A)–(E) are representative results from at least three independent experiments with at least two mice per time point.

Figure 6. Strong IL-2 Signals Negatively Impact CD8⁺ T Cell Memory Formation without Impairing Secondary Expansion

(A) 2.5 × 10⁵ congenically marked P14 cells that had been differentiated in high (100 U/ml) or low (10 U/ml) concentrations of IL-2 were transferred in a 1:1 mix to the same recipient. Representative mice were analyzed for the presence of the transferred cells in the spleen or lung (data not shown) 18 hr and 35 days after transfer. (B) Thirty-five days after transfer, the presence of the transferred P14 cells was determined (pLNs, peripheral lymph nodes). FACS plots are gated on total CD8⁺ T cells. High (H) IL-2 (Thy1.2⁺); Low (L) IL-2 (Thy1.2⁻). (C) CD62L expression on transferred P14 cells in the blood. (D) Thirty-five days after P14 transfer, recipient mice were infected with LCMV. Five days later, the frequency of the transferred P14s of total CD8⁺ T cells was determined in the liver and mesenteric lymph nodes (mLNs). (E) Absolute number of P14 T cells was determined in the spleens of representative mice before (day 35 after transfer) and after (day 5) LCMV infection. Data depicts average numbers ± SEM of at least two mice per time point per group. Panels (A)–(E) are representative results from two independent experiments.

Figure 7. Inflammatory Signals and IL-2 Signal Strength Induce Distinct Transcriptional Regimes

(A) The kinetics of mRNA expression in the presence and absence of inflammation. Naive P14 CD8⁺ T cells were stimulated by coculture with GP33 peptide (blue) and APCs, with or without CpG (red), for 2 days (d2) and then were recultured, with or without IL-12 (CpG/IL-12), in low (10 U/ml) or high (100 U/ml) IL-2. See also Figure S5. (B) The kinetics of surface IL-2Rα expression. P14 CD8⁺ T cells were primed with GP33 and APCs and cultured under the indicated conditions. IL-2Rα expression was determined by flow cytometry. (C) IL-2Rα expression prior to the first cell division. Naive P14 cells were loaded with CFSE and primed as shown in (B). After 36 hr, cells that had not diluted CFSE were gated, and IL-2Rα expression was determined by flow cytometry and is shown relative to naive cells stained prior to stimulation. (D) IL-2Rα expression in the presence of blocking αIL-2 antibody during priming. Cells were cultured as in (B).