Microbial short-chain fatty acids modulate CD8+ T cell responses and improve adoptive immunotherapy for cancer

Abstract

Emerging data demonstrate that the activity of immune cells can be modulated by microbial molecules. Here, we show that the short-chain fatty acids (SCFAs) pentanoate and butyrate enhance the anti-tumor activity of cytotoxic T lymphocytes (CTLs) and chimeric antigen receptor (CAR) T cells through metabolic and epigenetic reprograming. We show that in vitro treatment of CTLs and CAR T cells with pentanoate and butyrate increases the function of mTOR as a central cellular metabolic sensor, and inhibits class I histone deacetylase activity. This reprogramming results in elevated production of effector molecules such as CD25, IFN-γ and TNF-α, and significantly enhances the anti-tumor activity of antigen-specific CTLs and ROR1-targeting CAR T cells in syngeneic murine melanoma and pancreatic cancer models. Our data shed light onto microbial molecules that may be used for enhancing cellular anti-tumor immunity. Collectively, we identify pentanoate and butyrate as two SCFAs with therapeutic utility in the context of cellular cancer immunotherapy.

Fig. 1. Microbiota-derived SCFAs promote the production of effector cytokines in CTLs.

a The production of SCFAs, branched-chain fatty acids (BCFAs) and medium-chain fatty acids (MCFAs) by 16 human commensals was measured by GC-MS. All bacteria were grown in vitro until the stationary growth phase before the measurement of fatty acids in supernatants (n = 3 independent experiments). b The frequency of IFN-γ- and TNF-α-expressing CD8⁺ T cells cultured under suboptimal CTL conditions and stimulated with supernatant derived from M. massiliensis (1:40 or 1:20 supernatant-to-cell media ratios; n = 3 mice). c The percentage of IFN-γ⁺TNF-α⁺ CTLs, derived from SPF mice and treated with the indicated SCFAs for three days (n = 3 mice). d The secretion of TNF-α from CTLs treated with SCFAs was determined by ELISA (n = 3 mice). Statistical analysis was performed by two-tailed unpaired Student’s t-test; mean ± s.e.m. values are presented. Source data are provided as a Source data file.

Fig. 2. Pentanoate inhibits the class I HDAC enzymes and enhances the activity of mTOR in CD8⁺ T cells.

a Impact of bacterial SCFAs, BCFAs, and MCFAs on the activity of recombinant class I and II HDAC enzymes. TSA was used as a control pan-HDAC inhibitor. One of three independent experiments is shown. b The fluorogenic HDAC assay was applied to measure the HDAC inhibitory activity of SCFAs on CTLs. The value for unstimulated CTLs was arbitrarily set to 1.0. Four independent experiments were performed (analyzed by two-tailed unpaired Student’s t-test; data are shown as mean ± s.e.m). c–e Murine CD8⁺ T cells were polarized under suboptimal CTL-inducing conditions for three days. Representative contour plots (c) and bar graphs (d) indicate the frequency of IFN-γ⁺TNF-α⁺ cells and TNF-α secretion (e) after treatment with 300 nM mocetinostat (class I HDAC inhibitor) or 2.5 µM TMP-195 (class II HDAC inhibitor), respectively (n = 3 mice, analyzed by two-tailed unpaired Student’s t-test; data are shown as mean ± s.e.m). f Murine CTLs were cultured in medium containing 1.0% serum and treated with indicated HDACi for three days. Representative histogram plots and bar graphs indicate the phoshorylated levels of mTOR and S6 ribosomal protein, respectively (n = 3 mice, analyzed by two-tailed unpaired Student’s t-test; data are shown as mean ± s.e.m). Source data are provided as a Source data file.

Fig. 3. Pentanoate enhances anti-tumor activity of antigen-specific CTLs.

a Experimental design for the role of SCFAs in promoting anti-tumor immunity. b–g After three days of pretreatment with indicated HDAC inhibitors, CD8⁺CD45.1⁺ OVA-specific CTLs were transferred intraperitoneally (i.p.) into CD45.2⁺ mice bearing 5-days old B16OVA tumors. Tumor volume (b) and tumor mass (c) were analyzed on day 14 after inoculation of tumor cells (n = 6 mice/group combined from two independent experiments). The t-SNE plots in (d) show CD8⁺CD45.1⁺ cells among lymphocytes from the tumor-draining LNs. The percentage (e) and total cell number (f) of transferred CD8⁺CD45.1⁺ OT-I CTLs in the tumor-draining LNs at day 14 after tumor inoculation are shown. In g the total cell numbers of transferred antigen-specific IFN-γ⁺TNF-α⁺ CTLs were examined on day 14 after inoculation of B16OVA tumors in tumor-draining LNs (in e–g, n = 6 mice/group combined from two independent experiments). h–j OVA-specific CD45.1⁺ CTLs pretreated with pentanoate were adoptively transferred into CD45.2⁺ mice bearing 5-days old PancOVA tumors. Tumor volume (h) and tumor weight (i) were determined on day 23 post tumor inoculation. In j frequencies of transferred IFN-γ-producing CD8⁺ T cells in draining LNs and spleens were analyzed on day 23 post tumor inoculation (n = 6 mice/group combined from two independent experiments). Multiple group comparison (b, h) was performed by a linear-mixed effects model with Tukey correction. The two-tailed unpaired Student’s t-test was applied to compare two groups (n.s. = not significant; results are shown as mean ± s.e.m). Source data are provided as a Source data file.

Fig. 4. Pentanoate-treatment enhances the anti-tumor activity of murine CAR T cells.

a Experimental design for the analysis of SCFA-treated ROR1-specific murine CD8⁺ CAR T cells (CAR_ROR1). b The surface expression of CD25 on CAR T cells was measured by flow cytometry analysis after three days of stimulation with butyrate or pentanoate (n = 3 independent experiments). c, d The production of TNF-α and IFN-γ from CAR T cells was measured by ELISA (c) and flow cytometry analysis (d) after three days of stimulation with butyrate or pentanoate. Three similar experiments were performed. e–g Following the pretreatment with pentanoate for three days, ROR1-specific CAR T cells were transferred i.p. into mice bearing 5-days old PancROR1 tumors. Tumor volume (e) and tumor mass (f) were analyzed on day 14 post tumor inoculation (n = 6 mice/group combined from two independent experiments). The percentage and total cell number of transferred TNF-α⁺ and IFN-γ⁺ CAR T cells in the tumor tissue at day 14 after tumor inoculation are shown in (g). For all experiments, 0.75 mM butyrate and 2.5 mM pentanoate were used, respectively. Multiple group comparison in (e) was performed by a linear-mixed effects model with Tukey correction. In b–d and f, g, the two-tailed unpaired Student’s t-test was applied. Source data are provided as a Source data file.

Fig. 5. Pentanoate enhances the functional status of human CAR T cells.

a CD8⁺ T cells were isolated from peripheral blood of healthy donors were differentiated into CTLs in presence or absence of butyrate (1 mM), pentanoate (4 mM), mocetinostat (HDAC class I inhibitor, 300 nM) or TMP-195 (HDAC class II inhibitor, 2.5 µM). Representative contour plots (left) and graphs (right) show the frequency of IFN-γ⁺ TNF-α⁺ CD8⁺ T cells. (n = 4 combined from four independent experiments). b Experimental setup for the functional analysis of pentanoate-treated ROR1-specific human CD8⁺ CAR T cells (CAR_ROR1 T cells). c The surface expression of CD25 was measured following pentanoate treatment by flow cytometry. d The secretion of IL-2 was detected in supernatants of pentanoate-treated CAR_ROR1 T cells by ELISA. e The secretion of IFN-γ and TNF-α was analyzed in supernatants of pentanoate-treated CAR_ROR1 T cells by ELISA. f, g Proliferation of CAR_ROR1 T cells was determined by CFSE labeling (f). CAR_ROR1 T cells pretreated with pentanoate were stained with CFSE and subsequently co-cultured with K562^ROR1 cells in the absence of pentanoate. CD8⁺ T cells without the CAR construct were used as mock control cells. The cytolytic activity of CAR_ROR1 T cells was examined by analysis of specific lysis following encounter with luciferase-expressing K562^ROR1 cells (g). The percentage of lysed target cells was determined in 1 h intervals (effector-to-target cell (E:T) ratio = 2.5:1). Data points shown in the graphs represent CAR_ROR1 T cells derived from three different donors (c–g). Following pentanoate pretreatment, the stimulation was mediated by co-culture of CD8⁺ CAR_ROR1 T cells with ROR1-expressing K562 (K562^ROR1) cells in the absence of pentanoate. The groups in a and c–g were compared by two-tailed unpaired Student’s t-test, data shown as mean ± s.e.m. Source data are provided as a Source data file.