Lactic acid improves Treg manufacturing and in vivo function

Abstract

Adoptive cell therapy using regulatory T cells (Tregs) is a promising approach to suppress immune responses in autoimmunity and transplantation, but it is challenging to expand pure and optimally suppressive cells. Lactic acid (LA) is associated with enhanced Treg function in tumors so we hypothesized that it may be beneficial during Treg expansion. We found that addition of LA at day 3 post-stimulation onwards improved viability and purity, increased glycolysis upon re-stimulation, and led to superior suppressive function. In Tregs expressing chimeric antigen receptors (CARs) specific for HLA-A2, LA not only enhanced viability and purity but also significantly reduced tonic signaling-associated expression of exhaustion-associated markers (PD-1, TIM-3, LAG-3, TOX, and BLIMP-1). The effects of LA were not fully recapitulated by either pH-neutral lactate or low pH. In immunodeficient mouse models of chronic stimulation and xenogeneic graft-versus-host disease, LA-conditioned human Tregs demonstrated enhanced stability, reduced exhaustion marker expression, and improved efficacy. Thus, LA has a multimodal effect on human polyclonal and CAR Treg purity, viability, and function, representing a method to generate an optimal Treg product for cell therapy.

Keywords: autoimmunity; cell manufacture; exhaustion; lactate; lactic acid; metabolism; regulatory T cell; transplantation.

Graphical abstract

Figure 1

Lactic acid enhances Treg expansion and purity Naive human Tregs (CD4⁺CD45RO⁻CD45RA⁺CD127⁻CD25^hi) were stimulated using anti-CD3/CD28 Dynabeads and cultured for 9–15 days. At day 3, media was supplemented with 15 mM LA for the remainder of culture. (A) Schematic of Treg culture protocol. (B) Fold expansion and viability over time (n = 18). (C) Expression of FOXP3 and Helios at day 9 (n = 13). Representative figure shown on left. (D) Expression of CD25, CTLA-4, LAP, GARP, and CD39 at day 9 (n = 10). Connected lines indicate individual donor pairs. (E and F) Treg media was supplemented with 15 mM LA, 15 mM sodium lactate (SL), or HCl (pH 6.7) at day 3. (E) Fold expansion and viability over time (n = 9). (F) Expression of FOXP3 and Helios at day 9 (n = 12). Statistical analysis was carried out by two-way ANOVA with Sidak’s multiple comparisons test (B and E), paired t test (C, left and D), Wilcoxon matched-pairs signed rank test (C, right), or Friedman test with Dunn’s multiple comparison’s test (F). Colored asterisks indicate significant differences between control and LA (blue), SL (green), or HCl (purple). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 2

LA-conditioning enhances Treg suppressive function (A) Control or LA-conditioned human Tregs were stimulated for 24 h using anti-CD3/CD28 beads. Expression of ICOS, CTLA-4, PD-1, and 4-1BB was determined by flow cytometry (n = 6). Representative figures show unstimulated (dotted line) and stimulated (solid line) Tregs. Connected lines indicate paired donors. (B and C) Control or LA-conditioned Tregs were co-cultured with PBMCs and stimulated using anti-CD3/CD28 beads for 72 h. (B) Suppression of CD4 (left) and CD8 (right) T cell proliferation was determined by dye dilution (n = 18). (C) Expression of CD80 and CD86 was determined on B cells (n = 17). Representative figures show indicated cells in PBMC alone (black), control Treg (gray), and LA Treg (blue) conditions. Statistical analysis was carried out by paired T test (A) or two-way ANOVA with uncorrected Fisher’s LSD test (B and C). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

LA alters Treg metabolism Naive human Tregs were stimulated using anti-CD3/CD28 and cultured in control or LA-containing media for 9 days. (A and B) Glucose (A) and lactate concentration (B) was determined in media at 2-day intervals between D3-5, D5-7, and D7-9 (n = 3). Dotted line indicates concentration of glucose (A) or supplemented LA (15 mM) (B) in media at the beginning of culture. Net lactate production was calculated by subtracting 15 mM from the LA condition. (C and D) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured by Seahorse assay at day 9. (C) OCR over time (n = 3). (D) Basal OCR and ECAR (n = 3). (E) Control or LA-conditioned Tregs were re-stimulated at day 9 using anti-CD3/CD28 beads for 48 h in the absence of LA. Glucose and lactate concentration was determined in media (n = 3). Statistical analysis was carried out by two-way ANOVA with Sidak’s multiple comparisons test (A) or paired t test (D and E). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Figure 4

LA reduces CAR tonic signaling on Tregs Naive human Tregs were stimulated using anti-CD3/CD28 dynabeads, lentivirally transduced with HLA-A2-specific CARs with no co-stimulatory domain (A2.ζ) or with a CD28 (A2.28ζ), TNFR2 (A2.TNFRζ), or 4-1BB (A2.BBζ) domain. LA was supplemented to media from day 5 onwards. Cells were analyzed at day 12. (A) Schematic of CAR Treg transduction and expansion protocol. (B) CAR⁺ cell yield and viability (n = 8). (C) Cell-surface CAR expression, detected via the extracellular Myc tag (n = 5). (D) Proportion of FOXP3 and Helios expressing cells (n = 9). (E) Expression of FOXP3, CTLA-4, and LAP (n = 9). (F) Expression of Helios on FOXP3^pos cells relative to control-treated NGFR cells. Statistical analysis was carried out by two-way ANOVA with Fisher’s LSD test (B–E). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 5

LA reduces CAR Treg exhaustion marker expression Naive human Tregs were transduced with an HLA-A2-specific CAR (A2.ζ, A2.28ζ, A2.TNFRζ, or A2.BBζ) and cultured in control or LA-containing media. Cells were analyzed at day 12. (A) The proportion of CAR Tregs expressing 0–3 exhaustion markers (PD-1, LAG-3, and TIM-3) (n = 10). Asterisks indicate significant differences in proportion of cells between control and LA. (B) The expression of TOX and MFI of BLIMP-1 relative to NGFR-Tregs (n = 4–8). (C–E) CAR Tregs were co-cultured with HLA-A2⁺ dendritic cells (DCs) for 96 h. (C) Schematic of DC suppression assay (made with BioRender.com). (D) Expression of CD80 and CD86 on DCs after co-culture n = 6). (E) Expression of Ki67 and ICOS on CAR Tregs after co-culture (n = 5–6). Statistical analysis was carried out by two-way ANOVA with Sidak’s multiple comparisons test (A) or two-way ANOVA with Fisher’s LSD test (B, D, and E). Bars represent mean with individual donors connected by lines. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

LA-conditioning enhances CAR Treg purity and reduces expression of exhaustion markers under chronic stimulation in vivo LA-conditioned or control A2.28ζ-CAR Tregs were injected into HLA-A2⁺ NSG mice with autologous PBMCs at a 1:1 ratio. CAR Treg engraftment and phenotype was tracked weekly in blood. (A) Schematic of chronic stimulation model. (B) Absolute count of CAR⁺ cells in blood over time (n = 7). (C) Proportion CAR⁺ cells expressing FOXP3 and Helios in blood (n = 4–7). (D) Proportion of CAR⁺FOXP3^negHelios^neg cells expressing Ki67 in blood (n = 3–7). (E) Proportion of CAR⁺ cells in blood, liver, lung, and spleen at day 28 expressing 0–3 exhaustion markers (LAG-3, TIM-3, and PD-1). Statistical analysis was carried out by two-way ANOVA with Fisher’s LSD test (B–E). Schematic made using Biorender.com (A). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Figure 7

LA-conditioning enhances CAR Treg efficacy in vivo NSG mice were pre-conditioned with busulfan prior to injection of A2⁺ PBMCs with a sub-optimal ratio of LA-conditioned or control A2.28ζ-CAR Tregs (8:1 PBMC:CAR Treg). Mice were bled weekly and xenoGVHD scoring carried out at 1–3 day intervals. (A) Schematic of xenogeneic graft-versus-host disease (xenoGVHD) model. (B) Proportion of surviving mice over time (n = 8–9). (C) Absolute count of CAR⁺ cells in blood over time (n = 8–9). (D) Proportion of CAR⁺ cells expressing FOXP3 and Helios in blood (n = 8–9). (E) Correlation between the proportion of FOXP3^negHelios^neg cells at day 7 and survival. Statistical analysis was carried out by a Log rank (Mantel-Cox) test (B), two-way ANOVA with Fisher’s LSD test (C), or unpaired t test (D). Schematic made using Biorender.com (A). Data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.