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. 2009 Jul;15(7):808-13.
doi: 10.1038/nm.1982. Epub 2009 Jun 14.

Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells

Luca Gattinoni  1 , Xiao-Song Zhong, Douglas C Palmer, Yun Ji, Christian S Hinrichs, Zhiya Yu, Claudia Wrzesinski, Andrea Boni, Lydie Cassard, Lindsay M Garvin, Chrystal M Paulos, Pawel Muranski, Nicholas P Restifo
Affiliations

Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells

Luca Gattinoni et al. Nat Med. 2009 Jul.

Abstract

Self-renewing cell populations such as hematopoietic stem cells and memory B and T lymphocytes might be regulated by shared signaling pathways. The Wnt-beta-catenin pathway is an evolutionarily conserved pathway that promotes hematopoietic stem cell self-renewal and multipotency by limiting stem cell proliferation and differentiation, but its role in the generation and maintenance of memory T cells is unknown. We found that induction of Wnt-beta-catenin signaling by inhibitors of glycogen sythase kinase-3beta or the Wnt protein family member Wnt3a arrested CD8(+) T cell development into effector cells. By blocking T cell differentiation, Wnt signaling promoted the generation of CD44(low)CD62L(high)Sca-1(high)CD122(high)Bcl-2(high) self-renewing multipotent CD8(+) memory stem cells with proliferative and antitumor capacities exceeding those of central and effector memory T cell subsets. These findings reveal a key role for Wnt signaling in the maintenance of 'stemness' in mature memory CD8(+) T cells and have major implications for the design of new vaccination strategies and adoptive immunotherapies.

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Figures

Figure 1
Figure 1. TWS119 activates Wnt signaling in CD8+ T cells
Naive CD8+ T cells were primed in vitro with anti-CD3 (2 μg ml−1) and anti-CD28 (1 μg ml−1) specific antibodies with or without 7 μM TWS119. a, Western blot analysis of β-catenin and Gapdh in CD8+ T cells treated with or without TWS119. b, Electrophoretic mobility shift assay of nuclear extract from CD8+ T cells treated with or without TWS119 using P-labeled oligonucleotide probes designed from the TCF/LEF binding region of TCF1 target gene Fzd 7. Unlabeled oligonucleotide probes were used as competitor. c, Quantitative RT-PCR analysis of the expression of Tcf7, Lef1, Jun, Fzd7, and Nlk in CD8+ T cells treated with or without TWS119. Data are represented as mean +/− SEM. All data are representative of at least two independently performed experiments.
Figure 2
Figure 2. Wnt signaling inhibits CD8+ T cell proliferation and effector differentiation
a–c,CFSE-labeled naive pmel-1 CD8+ T cells were primed in vitro with CD8+ T cell depleted splenocytes pulsed with 1 μM hgp10025–33, in conjunction with 10 ng ml−1 IL-2 and titrated doses of TWS119. Four days following T cell activation, phenotypic (a) CFSE dilution assays (b) and cytokine and 51Cr release assays (c) were performed. Data in panel c are represented as mean +/− SEM. d, Quantitative RT-PCR analysis of the expression of Eomes in CD8+ T cells after priming with anti-CD3 and anti-CD28 specific antibodies with or without 7 μM TWS119. Data are represented as mean +/−SEM. e, Flow cytometry and enumeration of T cell subsets six days after vaccination with or without TWS119. WT mice received adoptive transfer of 106 naive pmel-1 thy1.1+ CD8+ T cells in conjunction with recombinant fowlpox-based hgp100 vaccine. Mice received four daily doses of TWS119 (at 30 mg kg−1) from day 0 to day 3 or DMSO as control. All data are representative of at least two independently performed experiments.
Figure 3
Figure 3. Wnt signaling promotes the generation of TSCM
CFSE-labeled, naive pmel-1 CD8+ T cells were primed in vitro with CD8+ T cell-depleted splenocytes pulsed with 1 μM hgp10025–33, in conjunction with 10 ng ml−1 IL-2 and 7 μM TWS119. a, Flow cytometry analysis of TWS119-treated and naive pmel-1 cells four days following T cell activation. b, Cytokine release assay of sorted CD44lowCD62Lhigh TWS119-treated and naive pmel-1 cells five days after antigenic stimulation. Data are represented as mean +/− SEM. c and d, CFSE dilution of sorted CD44lowCD62Lhigh TWS119-treated and naive thy 1.1+ (c) or ly5.1+ (d) pmel-1 cells one month after transfer into WT (c) and sublethally-irradiated WT, or Tcra−/−, or Rag1−/− mice (d). Data are shown on thy 1.1+ (c) or ly5.1+ (d) CD8+ lymphocytes. e, Flow cytometry analysis of sorted TWS119-treated and naive ly5.1+ pmel-1 cells one month after transfer into WT or B2m−/− mice. Data are shown on ly5.1+, CD8+ lymphocytes. f, Tumor treatment of myeloablated WT or B2m−/− mice bearing B16 tumors established for 7 days. Mice received age-matched, lineage-depleted bone marrow cells. On the following day, 106 CD44lowCD62Lhigh TWS119-treated or naive pmel-1 cells were transferred in conjunction with exogenous IL-2.g and h, Flow cytometry analysis of CFSE-labeled, sorted CD44lowCD62Lhigh TWS119-treated and naive ly5.1+ pmel-1 cells one month after transfer into sublethally-irradiated WT mice. Data are represented as the percentage of CD44lowCD62Lhigh cells as a function of CFSE dilution of two independent experiments (g) and as fraction of cells with any given number of divisions (h). All data are representative of at least two independently performed experiments.
Figure 4
Figure 4. TSCM possess enhanced in vivo recall response and anti-tumor activity compared to TCM and TEM
Pmel-1 naive CD8+ T cells were primed in vitro with splenocytes pulsed with 1 μM hgp10025–33, in conjunction with 10 ng ml−1 IL-2 with or without 7 μM TWS119. Five days after antigenic stimulation, TSCM, TCM or TEM were sorted based on the phenotype. Sublethally-irradiated WT mice received 5 × 104 pmel-1 TSCM, TCM or TEM in conjunction with a recombinant vaccinia virus encoding hgp100 and exogenous IL-2. a, Absolute numbers of adoptively transferred pmel-1 cells (identified by CD8+ thy1.1+ lymphocytes) in the spleens of treated animals. Data are represented as mean +/− SEM. b, Flow cytometry analysis for the expression of CD8 and thy1.1. c, Tumor treatment and survival of sublethally-irradiated WT mice bearing B16 tumors established for 10 days (n = 5 for all groups) receiving 4 × 104 pmel-1 TSCM, TCM or TEM in conjunction with a recombinant vaccinia virus encoding hgp100 and exogenous IL-2. Data are represented as mean +/− SEM. All data shown are representative of at least two independently performed experiments.
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References

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