Enhancing CD8 T-cell memory by modulating fatty acid metabolism

Abstract

CD8 T cells, which have a crucial role in immunity to infection and cancer, are maintained in constant numbers, but on antigen stimulation undergo a developmental program characterized by distinct phases encompassing the expansion and then contraction of antigen-specific effector (T(E)) populations, followed by the persistence of long-lived memory (T(M)) cells. Although this predictable pattern of CD8 T-cell responses is well established, the underlying cellular mechanisms regulating the transition to T(M) cells remain undefined. Here we show that tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an adaptor protein in the TNF-receptor and interleukin-1R/Toll-like receptor superfamily, regulates CD8 T(M)-cell development after infection by modulating fatty acid metabolism. We show that mice with a T-cell-specific deletion of TRAF6 mount robust CD8 T(E)-cell responses, but have a profound defect in their ability to generate T(M) cells that is characterized by the disappearance of antigen-specific cells in the weeks after primary immunization. Microarray analyses revealed that TRAF6-deficient CD8 T cells exhibit altered expression of genes that regulate fatty acid metabolism. Consistent with this, activated CD8 T cells lacking TRAF6 display defective AMP-activated kinase activation and mitochondrial fatty acid oxidation (FAO) in response to growth factor withdrawal. Administration of the anti-diabetic drug metformin restored FAO and CD8 T(M)-cell generation in the absence of TRAF6. This treatment also increased CD8 T(M) cells in wild-type mice, and consequently was able to considerably improve the efficacy of an experimental anti-cancer vaccine.

Figure 1. TRAF6-ΔT mice mount normal T_E responses, but have impaired T_M development following immunization with L. monocytogenes

Control (CTRL) and TRAF6-ΔT mice were LmOva-immunized and spleen (a and c) or spleen (SPL), lymph nodes (LN), and bone marrow (BM) cells (b) were Ova peptide-restimulated and analyzed for intracellular IFN-γ 7 days (a) (n=3–5 per group) or 60 days (n=3 per group) (b) post-infection. (c) 60 days post-immunization mice were challenged with LmOva and Ova-specific cells were analyzed 7 days post-challenge (n=3 per group). Dot plots and bar graphs show percentages of CD8 T cells producing IFN-γ (means ± standard deviation) *p=0.02 (b), 0.0005 (c).

Figure 2. TRAF6 intrinsically regulates CD8 T_M generation

OT-I cells (<5000) from OTI-TRAF6-WT and OTI-TRAF6-ΔT (CD45.2) (a–c) or CA-St5-OTI-TRAF6-WT and CA-St5-OTI-TRAF6-ΔT mice (CD45.2) (d,e) were transferred into CD45.1 recipients (n=5–9 per group) and LmOva-immunized. Three weeks post-transfer immune (a–d) or unimmunized mice (e) were LmOva-challenged. Mice were bled as indicated and cells surface stained. Dot plots show donor cells by CD45.2 and K^b/Ova tetramer (numbers indicate total CD8 T cell percentages that are host- or donor-derived). Line graphs represent percentages of donor-derived CD8 T cells (means ± standard deviation).

Figure 3. TRAF6-deficient CD8 T cells display defects in fatty acid metabolism that can be corrected by metformin

OT-I cells (<5000) from OTI-TRAF6-WT and OTI-TRAF6-ΔT mice (CD45.2) were transferred into CD45.1 recipients and immunized with LmOva. 6 (n= 3 per group) and 10 (n=5 per group) days post-infection donor cells were analyzed by microarray (a and SI Fig. 17). Tables generated using NIAID DAVID and KEGG databases (a). FAO (measured as mitochondrial β-oxidation) of activated OTI-TRAF6-WT and OTI-TRAF6-ΔT cells post-IL-2 withdrawal, *p value=0.012 (b). Ratio of β-oxidation to glycolysis in activated OTI-TRAF6-WT and OTI-TRAF6-ΔT cells post-IL-2 withdrawal (c). Western analysis of cells 16 hours post-IL-2 withdrawal +/− metformin (Acetyl CoA carboxylase (ACC) and Raptor are examined as targets of AMPK) (d). Mitochondrial β-oxidation of activated OTI-TRAF6-WT and OTI-TRAF6-ΔT cells +/− IL-2, where cells received nothing (−), triciribine (T), or metformin (M) for 16 hours, *p value = 0.003 (e). Western analysis of purified OTI-TRAF6-WT and OTI-TRAF6-ΔT T_E donor cells (7 days post-infection) (f).

Figure 4. Metformin treatment promotes T_M generation and protective immunity following infection and tumor challenge

(a,b) OT-I cells (<5000) from OTI-TRAF6-WT and OTI-TRAF6-ΔT mice (CD45.2) were transferred into CD45.1 recipients and immunized with LmOva. 8 days post-infection mice were injected daily with PBS (n=7–9 per group), metformin (n=7–9 per group), or rapamycin (n=5 per group) for three weeks and were then challenged with LmOva. Ova-specific responses of host and donor cells in the blood were measured 5 days post-challenge. Dot plot numbers reflect the percentages of total CD8 T cells that are host or donor-derived (Ova-specific). Bar graphs represent percent of CD8 T cells that are donor-derived (means ± standard error). *p values (comparing to PBS) = 0.015 (Met) and 0.000038 (Rap)(a), 0.0373 (Met) and 0.00028 (Rap)(b). (c) C57BL/6 mice were immunized and daily injections of metformin or PBS began 7 days post-infection. Three weeks later treatments ceased and mice were inoculated with EL4-Ova tumors. Tumors became palpable by 18 days post-inoculation and mice were euthanized when tumors reached 2cm. Graph reflects percent survival (n=9, metformin and n=8, PBS).