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. 2008 Jun 1;111(11):5326-33.
doi: 10.1182/blood-2007-09-113050. Epub 2008 Feb 14.

IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy

Christian S Hinrichs  1 , Rosanne Spolski, Chrystal M Paulos, Luca Gattinoni, Keith W Kerstann, Douglas C Palmer, Christopher A Klebanoff, Steven A Rosenberg, Warren J Leonard, Nicholas P Restifo
Affiliations

IL-2 and IL-21 confer opposing differentiation programs to CD8+ T cells for adoptive immunotherapy

Christian S Hinrichs et al. Blood. .

Abstract

IL-2 and IL-21 are closely related cytokines that might have arisen by gene duplication. Both cytokines promote the function of effector CD8(+) T cells, but their distinct effects on antigen-driven differentiation of naive CD8(+) T cells into effector CD8(+) T cells are not clearly understood. We found that antigen-induced expression of Eomesodermin (Eomes) and maturation of naive CD8(+) T cells into granzyme B- and CD44-expressing effector CD8(+) T cells was enhanced by IL-2, but, unexpectedly, suppressed by IL-21. Furthermore, IL-21 repressed expression of IL-2Ra and inhibited IL-2-mediated acquisition of a cytolytic CD8(+) T-cell phenotype. Despite its inhibitory effects, IL-21 did not induce anergy, but instead potently enhanced the capacity of cells to mediate tumor regression upon adoptive transfer. In contrast, IL-2 impaired the subsequent antitumor function of transferred cells. Gene expression studies revealed a distinct IL-21 program that was characterized phenotypically by increased expression of L-selectin and functionally by enhanced antitumor immunity that was not reversed by secondary in vitro stimulation with antigen and IL-2. Thus, the efficacy of CD8(+) T cells for adoptive immunotherapy can be influenced by opposing differentiation programs conferred by IL-2 and IL-21, a finding with important implications for the development of cellular cancer therapies.

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Figures

Figure 1
Figure 1
Antigen-induced acquisition of effector CD8+ T-cell phenotype is promoted by IL-2 and IL-15 but suppressed by IL-21. Naive pmel-1 Thy1.1 CD8+ T cells were primed with cognate antigen and the indicated cytokine for 3 days. (A-C) Flow cytometric determination of granzyme B, CD44, and IL-2Rα expression by Thy1.1 lymphocytes. (D) Real-time RT-PCR determination of Eomes expression 3 days after priming in 10 ng/mL of the indicated cytokine. Error bars indicate the standard error of the mean. (E) CFSE dilution 4 days after antigen priming of naive pmel-1 Thy1.1 CD8+ T cells with the indicated cytokine. Histograms are gated on Thy1.1 cells. The open histogram overlay indicates CFSE labeling prior to stimulation.
Figure 2
Figure 2
In contrast to IL-2 and IL-15, IL-21 does not promote acquisition of effector CD8+ T-cell function. (A,B) Pmel-1 CD8+ T cells were antigen-primed with 10 ng/mL of the indicated cytokine for 4 days, washed, and cocultured overnight with irradiated splenocytes pulsed with hgp10025-33. IFN-γ and IL-2 concentrations in the supernatants were determined by ELISA. (C) Chromium release cytolysis assay showing specific target killing by pmel-1 CD8+ T cells antigen-primed with 10 ng/mL of the indicated cytokine.
Figure 3
Figure 3
The antitumor efficacy of CD8+ T cells for adoptive transfer is impaired by IL-2 and IL-15, but enhanced by IL-21. Pmel-1 CD8+ T cells were primed with cognate antigen and 10 ng/mL of the specified cytokine for 4 days then adoptively transferred into tumor-bearing hosts. Vaccination and exogenous IL-2 were administered to mice in all but the “No treatment” group. (A) Tumor response to adoptive transfer of 2.5 × 105 cells. The cytokine present during priming is indicated. Error bars reflect the standard error of the mean. (B) Pmel-1 Thy1.1 CD8+ T cells were antigen-primed with 10 ng/mL of the indicated cytokine. At 4 days after priming, 5 × 105 cells per mouse were infused, and vaccine and IL-2 were administered. The number of Thy1.1 splenocytes was determined 12 days after transfer. The scatter plots indicate individual mice. The horizontal lines represent the means. *P < .05 compared with “No cytokine.”
Figure 4
Figure 4
IL-21 suppresses IL-2–induced effector CD8+ T-cell differentiation and substantially prevents IL-2–mediated impairment of CD8+ T cells for adoptive transfer. Naive pmel-1 CD8+ T cells were antigen-primed with 10 ng/mL IL-2, IL-21, or 10 ng/mL IL-2 combined with 10 ng/mL IL-21. (A) Eomes expression was determined by RT-PCR 3 days following priming. (B) Granzyme B expression was assessed by flow cytometry 3 days after priming. The mean fluorescence intensity is indicated. (C) Pmel-1 CD8+ T cells were primed with cognate antigen and 10 ng/mL of the indicated cytokine(s). After 4 days, 5 × 105 cells per mouse were adoptively transferred into tumor-bearing recipients. Vaccine and IL-2 were administered to all but the “No treatment” group. Tumor responses were assessed with serial measurements. Error bars represent the standard error of the mean.
Figure 5
Figure 5
Antigen priming with IL-21 imparts CD8+ T cells with a distinct gene expression program. Pmel-1 CD8+ T cells were antigen-primed with the cytokine indicated or without cytokine for 4 days, then restimulated for 4 hours without cytokine. Microarray gene expression analysis was then performed. Expression levels relative to the reference cells primed without cytokine are indicated. (A) Dendrogram showing the relatedness of gene expression patterns. (B) Microarray expression of selected genes associated with mature and immature effector CD8+ T cells. (C,D) Wild-type CD8+ T cells were stimulated with anti-CD3/anti-CD28 and 10 ng/mL of the indicated cytokine for 3 days and then restimulated in cytokine-neutral conditions for (C) 4 hours or (D) 3 days before real-time RT-PCR analysis. Expression of Tcf7 and Lef1 relative to β-actin is shown.
Figure 6
Figure 6
Antigen priming with IL-21 directs CD8+ T cells to express L-selectin during secondary stimulation. (A) Wild-type CD8+ T cells were anti-CD3/anti-CD28 stimulated with 10 ng/mL of the indicated cytokine for 3 days. Sell expression relative to β-actin expression was determined 4 hours after restimulation in cytokine-neutral conditions. (B) Four days after antigen priming with the indicated cytokine, pmel-1 Thy1.1 CD8+ T cells were restimulated with cognate antigen and 10 ng/mL IL-2 (regardless of the initial priming cytokine). Flow cytometry was performed 3 days after initiating the secondary response and histograms of L-selectin expression are displayed. The indicated cytokines and concentrations refer to the initial priming conditions. (C) Dot plots showing CFSE dilution and L-selectin expression by pmel-1 Thy1.1 CD8+ T cells that were antigen-primed with 100 ng/mL of the indicated cytokine for 4 days, labeled with CFSE, and restimulated with antigen and 10 ng/mL IL-2 (regardless of the cytokine present during initial priming). (D) Graphs of the mean fluorescence intensity of CFSE and L-selectin during restimulation. (E) Secondary expansion of cells antigen-primed with 10 ng/mL of the indicated cytokine for 4 days then restimulated with antigen and 10 ng/mL IL-2. Error bars indicate the standard error of the mean. (F) Tumor regression in response to adoptive transfer of 106 pmel-1 CD8+ T cells primed in 10 ng/mL of the indicated cytokine for 4 days, restimulated with antigen and IL-2 for 6 days, and then adoptively transferred. Vaccine and IL-2 were administered to mice in all except the “No treatment” group. Error bars reflect the standard error of the mean.
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