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. 2014 Jul 29;111(30):11115-20.
doi: 10.1073/pnas.1406259111. Epub 2014 Jul 14.

The tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) imposes a brake on antitumor activity of CD8 T cells

Marilyn Giordano  1 , Romain Roncagalli  1 , Pierre Bourdely  1 , Lionel Chasson  1 , Michel Buferne  1 , Sho Yamasaki  2 , Rudi Beyaert  3 , Geert van Loo  3 , Nathalie Auphan-Anezin  1 , Anne-Marie Schmitt-Verhulst  1 , Grégory Verdeil  4
Affiliations

The tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) imposes a brake on antitumor activity of CD8 T cells

Marilyn Giordano et al. Proc Natl Acad Sci U S A. .

Abstract

The transcription factor NF-κB is central to inflammatory signaling and activation of innate and adaptive immune responses. Activation of the NF-κB pathway is tightly controlled by several negative feedback mechanisms, including A20, an ubiquitin-modifying enzyme encoded by the tnfaip3 gene. Mice with selective deletion of A20 in myeloid, dendritic, or B cells recapitulate some human inflammatory pathology. As we observed high expression of A20 transcripts in dysfunctional CD8 T cells in an autochthonous melanoma, we analyzed the role of A20 in regulation of CD8 T-cell functions, using mice in which A20 was selectively deleted in mature conventional T cells. These mice developed lymphadenopathy and some organ infiltration by T cells but no splenomegaly and no detectable pathology. A20-deleted CD8 T cells had increased sensitivity to antigen stimulation with production of large amounts of IL-2 and IFNγ, correlated with sustained nuclear expression of NF-κB components reticuloendotheliosis oncogene c-Rel and p65. Overexpression of A20 by retroviral transduction of CD8 T cells dampened their intratumor accumulation and antitumor activity. In contrast, relief from the A20 brake in NF-κB activation in adoptively transferred antitumor CD8 T cells led to improved control of melanoma growth. Tumor-infiltrating A20-deleted CD8 T cells had enhanced production of IFNγ and TNFα and reduced expression of the inhibitory receptor programmed cell death 1. As manipulation of A20 expression in CD8 T cells did not result in pathologic manifestations in the mice, we propose it as a candidate to be targeted to increase antitumor efficiency of adoptive T-cell immunotherapy.

Keywords: T-cell activation; inflammation; tumor immunity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
T-cell–specific deletion of A20 in mice induces lymphadenopathy and infiltration of T cells in peripheral organs. (A–C) Total cell number (Left) or number of the indicated population (Right) per LN (A), spleen (B), or percentage of CD45+ cells among total cells in the liver (Left) and percentage of the indicated population among CD45+ cells (Right) in the liver (C) of maT-A20 mice (KO, open circles) or WT littermates (Litt., black circles). Each circle represents the number of cells from 1 mouse. Results from 6-, 9-, or 15-wk-old mice were pooled as we did not observe any significant variation between the various ages. Histograms represent pooled data from at least 10 mice per condition. (D) Percentage of CD62L− CD44+ T cells was determined by flow cytometry in LN, spleen (S), or liver (Liv.) from Littermate (Litt.) or maT-A20 mice (KO). (E) Hematoxilin/eosin labeling was performed on livers from 15-wk-old maT-A20 mice or littermate controls. Representative pictures from 6 individual mice are shown. (Scale bars, 100 µm.) (F) Fold increase of the indicated cytokine/chemokine in serum of maT-A20 mice (n = 12) versus littermate controls (n = 8) as measured by Luminex analysis.
Fig. 2.
Fig. 2.
CD8 T cells from maT-A20 mice have increased NF-κB activation upon stimulation. Purified CD8 T cells from maT-A20 mice (maT-A20) or littermates (Litt.) were stimulated with cross-linked anti-CD3 and anti-CD28 (A) or with coated anti-CD3 and soluble anti-CD28 (B and C) for the indicated time period. Cytoplasmic protein extracts (A) or cytoplasmic and nuclear protein extracts (C) were analyzed by Western blot for the indicated proteins. (B) Levels of IFNγ and IL-2 were measured by ELISA in the in vitro cultures used in C.
Fig. 3.
Fig. 3.
Increased cytokine production by CD8 T cells from maT-A20 mice. (A–C) CD8 T cells from LNs of P14-maT-A20 (gray line) or WT P14 littermate (black line) mice were left unstimulated (solid gray, A) or stimulated with GP33 peptide at a concentration ranging from 10−8 to 10−11 M and analyzed by flow cytometry for the indicated molecule. (A) Histograms represent the level of expression of the indicated molecules at 72 h postactivation (10−9 M GP33 peptide). (B) Supernatants from the P14-maT-A20 or WT P14 T cells stimulation with the indicated concentration of GP33 peptide were tested in triplicates by ELISA for content of IL-2 (24 h) or IFNγ (48 h). (A and B) Results are representative of three independent experiments. (C) Mean fluorescence intensity (MFI) of the indicated molecules was measured 48 or 72 h after the initial stimulation of WT P14 (black line) or P14-maT-A20 (gray line) CD8 T cells with the indicated concentration of peptide.
Fig. 4.
Fig. 4.
Inefficient tumor infiltration/elimination by CD8 T cells expressing high level A20. (A) CD8 T cells were sorted from melanomas that developed in TiRP mice (three independent samples). RNA levels of A20 from these cells (TILs) were compared with those from naïve CD8 T cells (N) and from in vitro activated CD8 T cells (Act) by quantitative RT-PCR. (B–F) The 5 × 104 sorted GFP+ TCRP1A-Luc CD8 T cells transduced with mock or A20 encoding retroviral constructs were injected in Rag−/−B10.D2 mice, injected with 106 P511 mastocytoma 7 d earlier. (B) P511 infiltration by TCRP1A-Luc T cells was monitored by bioluminescence. (C) At day 7, splenocytes and TILs were analyzed by flow cytometry for the indicated markers. Percentages of CD45+ CD8+ T cells are indicated on the dot plots. (D) TCRP1A CD8 T-cell numbers in TILs from three pooled experiments are shown. (E) Tumor growth was monitored every 2–3 d and pooled results from two independent experiments (10 mice per condition) are shown. (F) After sorting, CD45.1 mock-transduced and CD45.2 A20-transduced TCRP1A T cells were mixed at a 1:1 ratio and transferred in Rag−/−B10.D2 mice (hatched bars) or Rag−/−B10.D2 mice injected with 106 P511 mastocytoma 7 d earlier (empty white or gray bars). Mean percentage CD45.1+ or CD45.2+ among CD8 T cells at day 6 posttransfer in the LNs or in TILs (two experiments with 6 mice per condition) are shown.
Fig. 5.
Fig. 5.
P14 CD8 T cells from maT-A20 mice have increased antitumor potential. B16-GP33 cells (106) were injected s.c. in CD45.1 C57BL/6 mice and in vitro preactivated WT CD45.2 P14 or CD45.2 P14-maT-A20 CD8 T cells (6–8 × 106 cells) were transferred 7 d later. (A and B) Tumor growth was monitored with a caliper every 2–3 d. (C) At day 7 after transfer, levels of IFNγ and TNFα in mice sera were measured by ELISA. (D) On the same day, the number of CD45.2+ P14 T cells present in LNs and their proportion in TILs among total CD8 T cells was determined; number of CD45.2+ P14 T cells present in LNs of mice that received no tumor (no tumor) is also shown. (E) MFI of the indicated molecules was determined by flow cytometry on CD45.2+ P14 T cells and on endogenous CD45.1+ NK1.1+ cells (for GzmB only) present in the LNs or in TILs (T) directly ex vivo (PD-1 and GzmB) or after 4 h restimulation with GP33 peptide (IFNγ and TNFα). (A–E) Results are representative of two independent experiments. Numbers of mice are indicated in A and B and six mice per condition were used for C–E.
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