Dynamic Treg interactions with intratumoral APCs promote local CTL dysfunction

Abstract

Tregs control various functions of effector T cells; however, where and how Tregs exert their immunomodulatory effects remain poorly understood. Here we developed a murine model of adoptive T cell therapy and found that Tregs induce a dysfunctional state in tumor-infiltrating CTLs that resembles T cell exhaustion and is characterized by low expression of effector cytokines, inefficient cytotoxic granule release, and coexpression of coinhibitory receptors PD-1 and TIM-3. Induction of CTL dysfunction was an active process, requiring local TCR signals in tumor tissue. Tregs infiltrated tumors only subsequent to Ag-dependent activation and expansion in tumor-draining LNs; however, Tregs also required local Ag reencounter within tumor tissue to induce CTL dysfunction and prevent tumor rejection. Multiphoton intravital microscopy revealed that in contrast to CTLs, Tregs only rarely and briefly interrupted their migration in tumor tissue in an Ag-dependent manner and formed unstable tethering-interactions with CD11c+ APCs, coinciding with a marked reduction of CD80 and CD86 on APCs. Activation of CTLs by Treg-conditioned CD80/86lo DCs promoted enhanced expression of both TIM-3 and PD-1. Based on these data, we propose that Tregs locally change the costimulatory landscape in tumor tissue through transient, Ag-dependent interactions with APCs, thus inducing CTL dysfunction by altering the balance of costimulatory and coinhibitory signals these cells receive.

Figures

Figure 1. Tumor Ag–specific Tregs aggravate a state of dysfunction of tumor-infiltrating CTLs that resembles exhaustion.

(A) BALB/c mice were infused with GFP+ HA-Tregs and on the same day implanted in the flank with CT26HA tumors. (B–E) 9 days later, tumor-infiltrating, host-derived, polyclonal CTLs (B) were analyzed for their capacity to degranulate (C) and secrete IFN-γ (D) ex vivo in response to plate-bound CD3 Abs and for (E) expression of PD-1 and TIM-3, compared with CTLs from animals that did not receive HA-Tregs. (F) Frequency of cells in the indicated CTL subpopulations expressing IFN-γ upon restimulation. (G) Relative frequency of CTLs and CT26HA tumor cells (expressing the fluorescent fusion protein H2B-Cerulean) in day-9 tumors. (H) Thy1.2 mice received Thy1.1+ naive, HA-specific CL4 CD8+ T cells together with HA-Tregs, and expression of PD-1 and TIM-3 on tumor-infiltrating CL4 T cells was measured on day 9. Bar graphs in E and H show relative frequencies of CTLs expressing PD-1, TIM-3, both, or neither. Each experiment shown is representative of at least 2 with similar results; dashed lines in graphs in C and D indicate background in nonrestimulated CTLs; data represent n = 3–5 animals/group. All graphs indicate means; error bars denote SEM (E, F, and H). *P < 0.05.

Figure 2. Tregs rapidly amplify CTL dysfunction through local activity in tumor tissue.

(A) HA-Tregs prevented rejection of established CT26HA tumors by ex vivo–activated HA-CTLs injected at day 7. (B) HA-CTLs infiltrate both tumor stroma and parenchyma in presence of HA-Tregs. CT26HA nuclei were marked by expression of the blue fluorescent protein Cerulean fused to histone H2B. Tissue autofluorescence is depicted in red. Scale bar: 100 μm. (C) Expression of granzyme B and the lysosomal marker CD107a (intra- and extracellular) in tumor-infiltrating HA-CTLs 3 days after transfer into tumor-bearing mice harboring HA-Tregs or not. (D) Impaired degranulation and surface mobilization of CD107a in tumor-infiltrating HA-CTLs 2 days after transfer into animals harboring HA-Tregs. (E) Expression of PD-1 and TIM-3 by tumor-infiltrating HA-CTLs. Graph shows the frequency of HA-CTLs expressing PD-1, TIM-3, both, or neither, before and 2 days after transfer into mice harboring HA-Tregs or not. (F) Expression of effector cytokines upon short ex vivo restimulation of tumor-infiltrating HA-CTLs. Each experiment shown is representative of 2 (B–F) or 4 (A) with similar results (n = 3–5 per group). All graphs indicate means; error bars denote SEM (A and D–F). *P < 0.05.

Figure 3. Treg-dependent CTL dysfunction requires their local Ag recognition in tumor tissue.

(A) The HA515–523 determinant of HA was mutated in position 2 to generate HA Y516A and HA Y516D in order to prevent binding to H-2Kd while preserving the HA107–119 determinant. (B) CT26 cells expressing no HA, WT HA, or either mutant HA were mixed at 1:20 ratios with splenocytes from TCR-HA or CL4 TCR transgenic animals. T cell activation was measured 24 hours later as surface expression of CD69. Graph shows summary of data. Each analysis was performed in triplicate. (C) HA-Treg–seeded mice were implanted with both CT26HA and CT26HA Y516A tumors. When 5 × 106 HA-CTLs were injected at day 7, they would recognize HA only in CT26HA tumors, not CT26HA Y516A tumors, while HA-Tregs would encounter “their” HA determinant in both. (D) Similar HA-Treg recruitment in CT26HA and CT26HA Y516A tumors on day 11 after tumor implantation. (E) Expression of effector cytokines upon ex vivo restimulation of HA-CTLs from WT or mutant tumors retrieved 4 days after adoptive transfer into animals seeded with HA-Tregs or not. Graphs show fractions of CTLs expressing IFN-γ, TNF, both, or neither. (F) Expression of PD-1 and TIM-3 by HA-CTLs from WT or mutant tumors of animals harboring HA-Tregs or not. Graphs show fractions of CTLs expressing PD-1, TIM-3, both, or neither. Each experiment in C–F is representative of 2 (n = 3–4 per group) with similar results. All graphs indicate means; error bars denote SD (B) or SEM (D–F).

Figure 4. Tregs condition DCs to reduce their costimulatory activity and augment coexpression of PD-1 and TIM-3 by CTLs.

(A) LPS-activated splenic DCs were either not pulsed or pulsed with HA515–523 peptide, HA107–119 pepitde, or both and cocultured with HA-Tregs for 3 hours before addition of HA-CTLs, followed by analysis for expression of PD-1 and TIM-3 on CTLs 12 hours later. (B) As in A, but DCs were analyzed for expression of CD80 and CD86 directly before addition of CTLs. (C) HA-CTLs were reactivated for 3 hours by the indicated doses of anti-CD3ε antibodies in the absence or presence of activating anti-CD28 antibodies and analyzed for expression of PD-1 and TIM-3. (D) 10-day-old CT26HA tumors seeded with HA-Tregs or not were analyzed for expression of PD-L1, PD-L2, and galectin-9 on CD11b+CD11c+ DCs. (E) Expression of CD80 and CD86 on CD11b+CD11c+ DCs from 10-day-old CT26HA tumors harboring HA-Tregs or not. Each experiment shown is representative of at least 2 (n = 5 each) with similar results. Filled histograms show isotype staining. All graphs indicate means; error bars denote SD (A–C) or SEM (E). *P < 0.05.

Figure 5. Ag-dependent Treg dynamics in tumor tissue and dLNs.

(A) BALB/c mice were infused with GFP+ HA-Tregs and on the same day implanted in each flank with CT26HA and CT26 tumors. Inguinal dLNs were harvested from groups of 2–3 animals on various days after tumor implantation to analyze the number of HA-Tregs. (B) CFSE-labeled HA-Tregs in LNs were analyzed for proliferation and expression of activation markers CD44 and CD69 4 days after tumor implantation. Graph shows proportion of cells that had divided 1–4 and >4 times. FMO, fluorescence minus one control. (C) Tumors were harvested from groups of 2–3 animals on various days after tumor implantation to analyze the number of HA-Tregs. (D) As in A and C, but Thy1.1+ animals received a second infusion of DsRed-expressing HA-Tregs on day 3. Dot plot shows Thy1.2+ HA-Tregs in CT26HA dLNs on day 7. Graph shows proportion of HA-Tregs from first (GFP+) and second (DsRed+) infusion in dLNs, nondraining LNs (ndLN), spleen, and CT26HA tumors on day 7. (E) Expression of CD25 on HA-Tregs analyzed in C. Dot plots show results from day 7. Graph shows MFI of GFP+ HA-Tregs. Each experiment shown is representative of 2 (n = 3 per group) with similar results. All graphs indicate means; error bars denote SEM. *P < 0.05.

Figure 6. Treg-mediated inhibition of tumor rejection requires local Ag recognition in tumor tissue.

(A) The HA107–119 determinant of HA was mutated in position 5 to generate HA F111D and HA F111E in order to prevent epitope binding to I-Ed while preserving the HA515–523 determinant. (B) CT26 cells expressing either no HA, WT HA, or either mutant HA were mixed at 1:20 ratios with splenocytes from TCR-HA or CL4 TCR transgenic animals. T cell activation was measured 24 hours later as surface expression of CD69. Each analysis was performed in triplicate; graph shows summary of data. (C) Mice implanted with both CT26HA and CT26HA F111D tumors were repetitively injected with HA-Tregs on days 0, 4, and 8. HA-Tregs only expanded in CT26HA dLNs, but subsequently, after entry into the bloodstream, had access to both CT26HA and CT26HA F111D tumors. However, they would only re-encounter their cognate Ag in CT26HA tumors. (D) When 5 × 106 HA-CTLs were injected on day 7, HA-Tregs controlled HA-CTL–mediated rejection of CT26HA tumors, but not CT26HA F111D tumors. Data represent 3 mice per group in 1 representative of 2 independent experiments performed. All graphs indicate means; error bars denote SD (B) or SEM (D). *P < 0.05 vs. all other groups.

Figure 7. Tregs only transiently stabilize APC contacts in tumor parenchyma and stroma.

(A) DSFCs were installed on BALB/c mice; 2 days later, H2B-Cerulean–expressing CT26HA tumors were implanted into the chambers, and animals were infused with GFP+ HA-Tregs. On day 7, mice were injected with (unlabeled) HA-CTLs; 2 days later, HA-Treg behavior was recorded by MP-IVM. Micrograph shows HA-Tregs (green) at the border between tumor parenchyma (blue) and stroma 2 hours after injection of 150 kDa TRITC-Dextran, which was taken up by tumor-resident phagocytes (red). Right: Tracks of HA-Tregs with color-coded instantaneous velocity. (B) Similar to A, but no HA-Tregs were injected, and HA-CTLs expressed H2B-mRFP (green). Mice were i.v. injected with quantum dots to label the tumor vasculature (red). (C and D) Histograms of arrest coefficients of HA-Tregs (C) and HA-CTLs (D) in the tumor parenchyma and stroma of CT26 and CT26HA tumors. (E) Migratory tracks were smoothed through a moving average, and segments of continuous average motility <4 μm/min lasting >2 minutes were scored as arrests. (F) Duration of HA-Treg and HA-CTL arrests and frequency of arrests >5 minutes (boxed regions) in tumor parenchyma and stroma of CT26 and CT26HA tumors. Lines indicate medians. (G) Phenotype of CD45+ mCherry+ cells from CT26HA tumors. (H) 2 examples (of >20 observed) of HA-Tregs (green) interacting with CD11c-mCherry+ APCs (red) in a CT26HA tumor. Arrowhead indicates site of characteristic Treg tethering upon disengagement from APCs. Time shown in min:s. Data in C, D, and F are pooled from 5–8 individual recordings of different ROIs from 2–3 independent experiments per group. Data in G and H are from 2 independent experiments. Scale bars: 100 μm (A and B); 10 μm (H).

Figure 8. HA-Tregs and HA-CTLs colocalize, but do not directly interact, in tumor tissue.

DSFCs were installed on BALB/c mice; 2 days later, histone H2B-Cerulean–expressing CT26HA tumors (blue) were implanted into the chambers, and animals were infused with GFP+ HA-Tregs (green). On day 7, mice were injected with tdTomato-expressing HA-CTLs (red); 2 days later, the behavior of both populations in tumor tissue was recorded by MP-IVM. (A) Overview highlighting position of T cells in both tumor stroma and parenchyma. (B) 3 sample time series of HA-Tregs that migrated in close vicinity of HA-CTLs without forming a stable interface suggestive of a direct interaction. Time shown in min:s. Scale bars: 100 μm (A); 10 μm (B).