Metabolic support of tumour-infiltrating regulatory T cells by lactic acid

Abstract

Regulatory T (T_reg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells^1,2. Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME³, which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function^4-6. At the same time, T_reg cells maintain a strong suppression of effector T cells within the TME^7,8. As previous studies suggested that T_reg cells possess a distinct metabolic profile from effector T cells^9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral T_reg cells are linked. Here we show that T_reg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of T_reg cells in vitro. T_reg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. T_reg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in T_reg cells reveals that lactate uptake is dispensable for the function of peripheral T_reg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, T_reg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.

Extended Data Figure 1.. 1-amino-Cy5-glucose (GlucoseCy5) can act as a surrogate for 2NBDG in GFP/YFP T_reg reporter mice.

(a) Gating strategy for T_reg and T_conv cells (b) Representative plot of percent T_reg (CD4⁺Foxp3⁺) in the lymph node (LN) and tumor (TIL) of B16 bearing C57BL/6 mice day 14 post tumor inoculation. Representative plots gated on total CD4⁺ cells. (c) Glycolytic extracellular acidification rates (ECAR) of T_reg and T_conv cells sorted from LN and B16 TIL preparations as in (b) 48hrs after activation with αCD3/CD28 and IL-2. Oligo = oligomycin, 2-DG = 2-deoxy-D-glucose, ΔECAR = max reading after glucose minus basal ECAR. Max ECAR = max reading at after oligo minus basal ECAR. (d) Ex vivo 2NBDG uptake mean fluorescence intensity (MFI) by T_reg, T_conv, and CD8⁺ T cells from the LN and B16 TIL (**p=0.0045). (e) Ex vivo 2NBDG uptake by CD44⁺CD62L⁻ and CD44⁻CD62L⁺ T_reg and T_conv cells isolated from the LN of Foxp3-Ametrine reporter mice. (f) Lymphocytes from Foxp3-Ametrine reporter mice were simultaneously pulsed with 2NBDG and GlucoseCy5. Representative plot is gated CD4⁺ Foxp3⁺ with tabulation of percent GlucoseCy5⁺ of 2NBDG⁺ T_reg. (g) Ex vivo GlucoseCy5 uptake by CD44⁺ T_reg and T_conv cells from the LN and B16 TIL. Representative plots gated on CD44⁺ CD4⁺ cells (**p=0.0025). (h) GlucoseCy5 positivity in Nrp1 negative and positive T_reg cells. (i) Ex vivo GlucoseCy5 uptake by T_conv cells isolated from various tissues. Results are representative of three (a-d,f-i) or two (e) independent experiments. Significance (*p< 0.05, **p <0.01, ***p < 0.001, ****p<0.0001) was determined by paired two-tailed t test (e), unpaired two-tailed t test (d,f,g,h) or two-way ANOVA with Sidak's multiple comparisons test (c). Data are mean values of biological replicates ±SEM.

Extended Data Figure 2.. Glucose avid T_reg cells harbor a weaker T_reg cell signature but retain viability and some suppressor activity.

(a) T_reg cells were sorted based on GlucoseCy5 uptake and assayed for their ability to suppress the proliferation of CellTrace Violet (CTV) labeled T_conv cells at 1:4 (Treg:Tconv) (LN*p=0.02, TIL *p=0.041). (b) Experimental diagram for (c, d). (c) Representative histogram and quantification of viability of sorted 2NBDG^hi and 2NBDG^lo T_reg cells after 72hrs in a suppression assay. (d) 2NBDG uptake by 2NBDG^hi or 2NBDG^lo T_reg as in (b). (e) Representative histograms and tabulation of T_reg signature gene expression between 2NBDG low and high T_reg cell subsets (Nrp1 **p=0.0016, CD73 *p=0.029 **p=0.0076, TIGIT LN*p=0.04 TIL*p=0.016, CD25 *p=0.037). (f) Weights of Rag1^−/− mice that received an adoptive transfer of either 2NBDG^lo or 2NBDG^hi T_reg cells plus Thy1.1⁺ T_conv cells I.V. (g) Representative histogram of T_conv responder cell proliferation after 72hrs of co-culture with T_reg cells (1:4) conditioned in 0, 5, or 25mM glucose for 3 days. The suppression assay occurred in 25 mM glucose conditions. (h) Geneset enrichment plot of cellular lipid catabolic process from TIL 2NBDG^hi vs 2NBDG^lo T_reg cells. (i) mRNA expression of Slc16a1 and Ldha in LN- and B16-infiltrating T_reg and T_conv cells by qPCR (Ldha **p=0.004, Slc16a1 **p=0.009). (j) Slc16a1 and Ldha mRNA expression in T_reg or T_conv cells activated overnight and conditioned in the glucose concentration indicated for 3 days (normalized to 25mM glucose, significance between T_reg and T_conv) (Ldha **p=0.002 Slc16a1 5 *p=0.030, 0 *p=0.046). Results are representative of three (a,e,h,i,j), or two (c,d,f) independent experiments. Significance (*p< 0.05, **p <0.01, ***p < 0.001) determined by unpaired two-tailed t test (a,f,i,j) or paired two-tailed t test (c,d) or two-way ANOVA with Sidak’s multiple comparisons test (e). Data are mean values of biological replicates ±SEM.

Extended Data Figure 3.. T_reg cells are resistant to lactic acid and use PEPCK-mediated metabolic pathways to support their proliferation.

(a) Proliferation of CD8⁺, T_conv, and T_reg cells labeled with CellTrace Violet (CTV) activated in media with lactic acid for 3 days (**p=0.0058). (b) IFN-γ production of CD8 and T_conv cells conditioned as in (a) then restimulated overnight with PMA/ionomycin (CD8 **p=0.0042, Tconv *p=0.02). (c) Suppression assay using T_reg cells conditioned as in (a) performed in absence of additional lactic acid. (d) Representative histogram and quantification of pHrodo⁺ T_reg cells taking up 2NBDG. (e) Representative flow plot of Ki67 expression by B16-infiltrating T_reg cells from mice treated ±3MP for 3 days. (f) Suppressive capacity of T_reg cells, isolated from mice as in Fig.3k. (g) Percent Foxp3⁺ cells, T_conv:T_reg ratio, and CD8:T_reg ratio within the TIL of mice treated as in (f). (h) Tumor growth curve of B16 in C57BL/6 mice treated with H₂O or 3MP. (i) Proliferation of T_reg and T_conv cells activated and cultured in 10mM lactic acid media ±3MP for 3 days (*p=0.016). (j) Capacity of activated T_reg cells conditioned in 10mM lactic acid media ± 250μM 3MP for 3 days to suppress the proliferation of CTV labelled T_conv cells. (k) IFN-γ expression by CD8 and T_conv cells in Foxp3^Cre or Slc16a1^f/fFoxp3^Cre mice treated as in (f). (l) Percent Foxp3⁺ cells, T_conv to T_reg ratio, and CD8 to T_reg ratio within LN and tumor of Slc16a1^f/fFoxp3^Cre mice treated as in (f). Results are representative of four (d), three (a,b,c,f,g,i), or two (h,j,k,l) independent experiments. Significance (*p< 0.05, **p <0.01, ***p < 0.001, ****p<0.0001) determined by unpaired two-tailed t test (d,e,g,i,k,l) or one-way ANOVA with Dunnett’s multiple comparisons test (a,b) or two-way ANOVA with Sidak's multiple comparisons test (c,f,h,j). Data are mean values of biological replicates ±SEM.

Extended Data Figure 4.. MCT1 is efficiently deleted in T_reg cells of Slc16a1^f/fFoxp3^YFP-Cre mice and is not required for peripheral T_reg cell function.

(a) Slc16a1 expression in T_conv and T_reg cells from the lymph nodes (LN) of Foxp3^Cre or Slc16a1^f/fFoxp3^Cre mice. (b) T_reg cell mRNA expression of Slc16a3 (MCT4) from mice as in (a). CD44, Ki67, and IFN-γ production by LN CD8⁺ T cells (c, *p=0.039) or T_conv cells (d, **p=0.007) from mice as in (a). (e) Capacity of LN-derived T_reg cells as in (a) to suppress proliferation of CellTrace Violet (CTV) labelled T_conv cells. (f) Percent Foxp3⁺ and Nrp1⁺Helios⁺ LN T_reg cells from mice as in (a) (*p=0.013). (g) CD44, Ki67, and CD62L expression by LN T_reg cells as in (a). (h) CD39 and CD73 expression by LN T_reg cells as in (a). Percent of Foxp3⁺ cells (i) or GlucoseCy5⁺ T_reg cells (j) from various tissues of mice as in (a) (thymus *p=0.014, B16 *p=0.019). (k) Normalized ear thickness of imiquimod treated mice as in (a). (l) Percent marker positive T_reg cells from mice treated as in (k). (m) Foxp3 expression in transferred wild type or Slc16a1-deficient T_reg infused with Thy1.1⁺ T_conv cells into Rag1^−/− mice iv. (*p=0.02) (n) Weight of Rag1^−/− mice over time from (m). (o) Representative sections of the colon and quantified histology scores 7 weeks post transfer from mice as in (m) and Fig.2d (**p=0.001, **p=0.009). (p) Anti-pimonidazole area over tumor area (B16) calculated from immunofluorescence from Foxp3^Cre and Slc16a1^f/fFoxp3^Cre mice. (q) IL-2 and IFN-γ expression by Slc16a1-deficient T_conv cells stimulated overnight with PMA/ionomycin. Results are representative of four (c,d,f,g), three (a,b,e,h,j,p,q), or two (k,l,m,i,o) independent experiments. Significance (*p< 0.05, **p <0.01, ****p < 0.0001) determined by unpaired two-tailed t test (a-d,f-q) or two-way ANOVA (e). Data presented as mean values of biological replicates ±SEM.

Extended Data Figure 5.. Acute deletion of MCT1 results in similar immunologic phenotypes in B16 melanoma and predilection towards a fragile T_reg cell phenotype.

(a) Foxp3^Cre-ERT2 and Slc16a1^f/fFoxp3^Cre-ERT2 mice were treated 5 consecutive days with tamoxifen I.P. prior to inoculation with 1.5 x 10⁵ B16 tumor cells. Following inoculation, tamoxifen was administered I.P. 3 times a week until sacrifice at day 14. (b) Percent Foxp3⁺ CD4⁺ cells in the lymph node (LN) and tumor (TIL) as in (a) (**p=0.005). (c) Tabulation of IFN-γ production by T_reg cells from the LN and TIL of mice as in (a) (**p=0.002). (d) Glucose consumption by T_reg cells from the TIL of mice as in (a). (e) Percent proliferating T_conv cells from the TIL of mice as in (a) (*p=0.037). (f) Representative flow plot and tabulation of IFN-γ and TNFα production by CD8 T cells from the TIL of mice as in (a) (**p=0.003, *p=0.02). Tabulation of IFN-γ production by T_conv cells from the TIL of mice as in (a) (**p=0.003). (g) Nrp1 mean fluorescence intensity on MEER intratumoral Treg cells derived from Foxp3^Cre-ERT2 and Slc16a1^f/fFoxp3^Cre-ERT2 mice treated as in (a) (*p=0.034). (h) Tim-3 expression by MEER derived CD8 T cells from mice as in (g) (**p=0.003). Results are representative of four (b,c,d,e), or three (f), or two (g,h) independent experiments. Significance (*p< 0.05, **p <0.01) determined by unpaired two-tailed t test (b-h). Data presented as mean values of biological replicates ±SEM.

Figure 1.. Regulatory T cells possess a distinct metabolic profile from conventional T cells in normal and transformed tissues.

(a) (left) Representative histogram of T_conv responder cell proliferation dye dilution after 72hrs of co-culture with T_reg cells isolated from B16, Cl24 (melanoma), or MEER (HPV+ HNSCC) tumors. (right) Capacity of T_reg cells to suppress the proliferation of CellTrace Violet (CTV) labelled T_conv cells as a function of the glycolytic extracellular acidification rate (ECAR) of the tumors from which they were isolated (*p=0.047). (b) Percent T_reg cells (CD4⁺ Foxp3⁺) of total CD4⁺ T cells from lymph node (LN) and tumor infiltrating lymphocyte (TIL) preparations from C57BL/6 mice bearing B16 melanoma tumors (14 d post intradermal injection) (c) Percent proliferating (Ki67⁺) T_reg and T_conv cells (CD4⁺ Foxp3^-) from mice as in (b). (d) ECAR of in-Seahorse activated T_reg, CD8+, CD44+CD62L− and CD44-CD62L+ T_conv cells sorted from LN and spleen of Foxp3 reporter mice. Oligo = oligomycin, 2-DG = 2-deoxy-D-glucose, ΔECAR = max reading after αCD3 minus basal ECAR. (*p=0.022) (e) Flow cytogram depicting ex vivo 2NBDG uptake by T_reg and T_conv cells from the LN and B16 TIL of Foxp3 reporter mice. Representative plots gated on CD4+ cells. (**p=0.006) (f) Diagram of experimental procedure and quantification of ex vivo 2NBDG uptake by CD44⁺ OT-II T_reg and T_conv cells isolated 5 days after transfer into congenically mismatched hosts infection with Vaccinia-OVA. (g) Representative histogram and quantification of ex vivo GlucoseCy5 uptake by T_reg cells isolated from various tissues (*p=0.047). Results are representative of five (b), three (a,c,e, and g) or two (d,f) independent experiments. (*p< 0.05, **p <0.01, ***p < 0.001 ****p<0.0001) by unpaired two-tailed t test (a,b,d,f,g) or two-way ANOVA with Sidak’s multiple comparisons test (c,e). Data are mean values of biological replicates ±SEM.

Figure 2.. Glucose avidity is associated with reduced T_reg cell functional identity.

(a) Flow cytogram of CD4+ T cells from B16-bearing Foxp3^Ametrine mice depicting sorting strategies for T_reg cells based on their glucose uptake. T_reg cells sorted based on 2NBDG uptake were assayed for their ability to suppress the proliferation of CellTrace Violet (CTV) labeled T_conv cells. Representative histogram shows 1:4 ratio of T_reg:T_conv. (**p=0.0056). (b) T_reg cell signature gene expression in RNAseq analysis of 2NBDG^hi or 2NBDG^lo T_reg cells sorted from lymph node (LN) or tumor (TIL) of B16 melanoma-bearing Foxp3 reporter mice as in (a). (c) Production of IL-10, IFN-γ, and IL-2 by sorted 2NBDG^lo and 2NBDG^hi T_reg cells stimulated overnight with PMA/ionomycin and stained intracellularly (*p=0.02). (d) Diagram and colon histology Rag1^−/− mice that received either 2NBDG^lo or 2NBDG^hi Thy1.2⁺ T_reg cells plus Thy1.1⁺ T_conv cells I.V and followed for 7 wks. (e) Percent of transferred 2NBDG^lo or 2NBDG^hi T_reg cells as in (d) expressing Foxp3 within the spleen (Spln), mesenteric lymph node (mLN), intraepithelial layer (IEL), and lamina propria (LP) (*p=0.02, **p=0.004). (f) Representative histogram and tabulation of GlucoseCy5 uptake by T_reg cells isolated from the non-draining lymph node (ndLN), pancreatic lymph node (pLN), and the islet of 10-12-week-old NOD.Foxp3^GFP-Cre mice (**p=0.005). (g) (left) Diagram of experimental procedure and (right) capacity of T_reg cells conditioned in 0,5, or 25mM glucose media to suppress the proliferation of CellTrace Violet (CTV) labelled T_conv cells. (1:4 *p=0.031, 1:8 *p=0.012 between 0 and 25 mM). (h) Glycolytic pathway gene expression as in (b). Results are representative of three (a,b,c,g,h), or two (d,e,f) independent experiments. Significance (*p< 0.05, **p <0.01, ***p < 0.001, ****p<0.0001) was determined by unpaired two-tailed t test (a,c,d,e,f), or two-way ANOVA with Tukey’s multiple comparison test (g). Data are mean values of biological replicates ±SEM.

Figure 3.. T_reg cells metabolize lactic acid to support their proliferation and suppressor function.

(a) Lactate concentration in spleen or B16 melanoma interstitial fluid (*p=0.041). (b) Lymphocytes from Foxp3^Cre mice were loaded with pHrodo Red (intracellular pH dye) and pulsed with 5mM lactic acid. Results are change of MFI from t=0 (**p=0.0012). (c) Lactic acid uptake determined as in (b) by T_reg cells infiltrating various tissues. (d) Lactic acid uptake from T_reg and T_conv cells isolated from islets, non-draining (ndLN), and pancreatic lymph node (pLN) of 10-12-week-old NOD.Foxp3^GFP-Cre mice (*p=0.017, **p=0.001). (e) Flow cytogram and tabulation depicting Nrp1 and CD44 expression in T_reg cells based on lactate-elicited pH change (*p=0.018, **p=0.0097). (f) Diagram showing incorporation of ¹³C derived from lactate into downstream metabolites. (g) Relative abundance determined by mass spectrometry of intracellular lactate, pyruvate, malate, and citrate in T_reg and T_conv cells activated overnight then pulsed with uniformly labeled ¹³C-lactate (m+n equal to the number of incorporated heavy carbons) (**p=0.0025). (h) Relative abundance of PEP derived from ¹³C-lactate as in (g) (*p=0.036, **p=0.0011). (i) Capacity of T_reg cells conditioned for 3 days in 25mM glucose media ±10mM lactic acid to suppress the proliferation of CellTrace Violet (CTV) labelled T_conv cells (*p=0.021). (j) As in (h) with the addition of PEPCK inhibitor 3MP (*p=0.018). (k) Ki67 expression in intratumoral T_reg cells of B16 tumor-bearing mice treated with 3MP or water for 3 days (*p=0.015). (l) IFN-γ expression by B16-infiltrating CD8⁺ T cells from mice treated as in (k) for 5 days. Results are representative of three (a,c,e,g,h,k), or two (b,d,i,j,l) independent experiments. Significance (*p< 0.05, **p <0.01, ***p < 0.001, ****p<0.0001) determined by unpaired two-tailed t test (a,b,d,e,i,j,k,l) or unpaired one-tail t test (l) or two-way ANOVA with Sidak's multiple comparisons test (g,h). Data are mean values of biological replicates ±SEM.

Figure 4.. Tumor-infiltrating T_reg cells require lactate uptake to maintain their high suppressive function.

(a) Lactate-elicited pHrodo fluorescence in Foxp3^Cre or Slc16a1^f/fFoxp3^Cre T_reg cells (*p=0.041). (b) B16 tumor growth and survival of Foxp3^Cre and Slc16a1^f/fFoxp3^Cre mice (*p=0.0235) (c) Percent of B16-infiltrating PD-1⁺Tim-3⁺ CD8⁺ T cells day 14 post tumor injection in mice as in (a) (*p=0.017). (d) Proliferation of tumor infiltrating T_conv and CD8⁺ T cells as in (c) (**p=0.009). (e) Tumor-infiltrating CD8⁺ and T_conv cells restimulated with PMA/ionomycin and stained for IFN-γ (*p=0.039). (f) Capacity of WT or Slc16a1-deficient tumor-infiltrating T_reg cells to suppress proliferation of CTV labeled T_conv cells. (g) Percent proliferating tumor infiltrating T_reg cells from mice as in (c). (*p=0.011). (h) Percent or MFI of WT and Slc16a1-deficient T_reg cells expressing Nrp1, PD-1, CD44 from B16 tumors (TIL) as in (c) (*p=0.029, **p=0.009). (i) GlucoseCy5 uptake by B16 tumor-infiltrating T_reg cells as in (c) (j) Pimonidazole staining of WT and Slc16a1-deficient T_reg cells from B16 tumors as in (c) (*p=0.029). (k) Tumor growth of B16 (melanoma, **p=0.0011) MC38 (adenocarcinoma, *p=0.023), and MEER (HNSCC, *p=0.0146) in tamoxifen treated Foxp3^Cre-ERT2 and Slc16a1^f/fFoxp3^Cre-ERT2 mice. Mice were given tamoxifen 5 consecutive days prior to tumor injection then 3x per week. (l) Survival of tamoxifen treated Foxp3^Cre-ERT2 and Slc16a1^f/fFoxp3^Cre-ERT2 mice injected with B16 and treated with IgG or anti-PD-1 antibodies 3x weekly (**p=0.002). Results are representative of four (b,c,d,f,g,h(Nrp1, CD44),i,j), three (a, k(B16)), or two (h(PD-1), k(MC38, MEER),l) independent experiments. Significance (*p< 0.05, **p <0.01, ***p < 0.001, ****p<0.0001) determined by the log-rank test for survival curves (b,l) or unpaired two-tailed t test (a,c,d,e,g,h,i,j) or two-way ANOVA (b,f,k). Data are mean values of biological replicates ±SEM.