T cells expressing chimeric antigen receptor promote immune tolerance

Abstract

Cellular therapies based on permanent genetic modification of conventional T cells have emerged as a promising strategy for cancer. However, it remains unknown if modification of T cell subsets, such as Tregs, could be useful in other settings, such as allograft transplantation. Here, we use a modular system based on a chimeric antigen receptor (CAR) that binds covalently modified mAbs to control Treg activation in vivo. Transient expression of this mAb-directed CAR (mAbCAR) in Tregs permitted Treg targeting to specific tissue sites and mitigated allograft responses, such as graft-versus-host disease. mAbCAR Tregs targeted to MHC class I proteins on allografts prolonged islet allograft survival and also prolonged the survival of secondary skin grafts specifically matched to the original islet allograft. Thus, transient genetic modification to produce mAbCAR T cells led to durable immune modulation, suggesting therapeutic targeting strategies for controlling alloreactivity in settings such as organ or tissue transplantation.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. mAbCAR construct and its expression in transfected T cells and Tregs.

(A) mAbCAR construct scheme. Schematic representation of mAbCAR construct expressing the anti-FITC scFv (1X9Q) and intramembrane CD28 and CD3ζ stimulatory domains. (B) mAbCAR protein model. Model of mAbCAR molecule expression by transfected T cells and loading with FITC-conjugated mAb. (C) mAbCAR expression in T cells. Representative flow cytometry plots of mAbCAR expression by untransfected or transfected T cells measured by anti-FLAG and different FITC-conjugated mAbs. (D) mAbCAR expression in Tregs. Representative flow cytometry plots of mAbCAR expression by untransfected or transfected Tregs measured by anti-FLAG and different FITC-conjugated mAbs. (E) mAbCAR expression over time. Kinetic of mAbCAR expression over time in T cells after transient transfection and incubation in vitro, assessed by flow cytometry Data derive from 1 of 3 consecutive experiments; mean ± SEM.

Figure 2. mAbCAR-expressing T cells are activated by FITC binding.

(A) Analysis of mAbCAR-expressing CD4+ T cells. Whiskers plots represent the percentage of surface expression of selected markers measured by flow cytometry (CD69, LAG3, PD1) before or after in vitro exposure to FITC (24 hours) in untransfected CD4+ T cells (red), CD4+ T cells that were mock transfected (black), and CD4+ T cells that were transfected with mAbCAR construct (gray). Reported results derive from 3 independent experiments. Two-tailed Student’s t test; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001. (B) Activation of mAbCAR T cells in vitro is target specific. mAbCAR T cells loaded with FITC-isotype control mAb or FITC-anti-MAdCAM1 mAb and cultured for 1 day with irradiated cell suspension derived from syngeneic spleen where MAdCAM1 was also expressed. CD25 and CD69 surface expression was measured by flow cytometry. Reported results derive from 3 independent experiments. Two-tailed Student’s t test; mean ± SEM; *P < 0.05; **P < 0.01. (C) FITC mAbCAR does not modify T cell distribution. Percentage of naive (CD62L+CD44neg), central memory (CD62L+CD44+), and effector memory (CD62LnegCD44+) assessed by flow cytometry from mAbCAR T cells cultured as in B. Reported results derive from 3 independent experiments. Two-tailed Student’s t test; mean ± SEM. (D) Representative flow cytometric plot of naive (CD62L+CD44neg), central memory (CD62L+CD44+), and effector memory (CD62LnegCD44+) in vitro–cultured FITC-isotype or FITC-MAdCAM1-mAbCAR T cells.

Figure 3. Tissue-specific FITC mAbs modulate mAbCAR T cell homing and function in vivo.

(A) Targeting mAbCAR T cells regulates homing. The graph shows bioluminescent signal from allogeneic luc+ untransfected Tcons (black), luc+ SDF1-mAbCAR Tcons (blue), or luc+ MAdCAM1-mAbCAR Tcons (red) at day +4, +7, and +12 after transfer in mice that received TCD BM at day 0. One representative of three consecutive experiments is presented; 2–4 mice/group were used in each experiment. ANOVA test with Bonferroni post-test; mean ± SEM; ***P < 0.001. (B) Targeting mAbCAR T cells alters localization. Representative bioluminescent images of mice that received luc+ untransfected Tcons, luc+ SDF1-mAbCAR Tcons, or luc+ MAdCAM1-mAbCAR Tcons at day +12 after transfer. Data are representative of 1 of 3 consecutive experiments. (C) Targeting mAbCAR T cells modulates GvHD. GvHD score over time of recipient mice that received allogeneic untransfected Tcons, SDF1-mAbCAR Tcons, or MAdCAM1-mAbCAR Tcons. One representative of three independent experiments is presented. ANOVA test with Bonferroni post-test; mean ± SEM; ***P < 0.001. (D) mAbCAR T cells mediate graft-versus-tumor effects. Tumor growth analyzed by BLI in lethally irradiated BALB/c mice that received allogeneic C57BL/6 TCD BM and luc+ A20 alone (black), luc+ A20 and allogeneic isotype-mAbCAR Tcons (gray), or luc+ A20 and allogeneic SDF1-mAbCAR Tcons (blue). One representative of two consecutive experiments is reported; at least 3 mice/group were used. Two-tailed Student’s t test; mean ± SEM; ***P < 0.001. (E) mAbCAR T cells eliminate tumor in vivo. Representative bioluminescent images of lethally irradiated BALB/c mice that received allogeneic C57BL/6 TCD BM and luc+ A20 alone, luc+ A20 and isotype-mAbCAR Tcons, or luc+ A20 and SDF1-mAbCAR Tcons at day +7, +14, and +21 after transplant. One representative of two consecutive experiments is reported; at least 3 mice/group were used. (F) SDF1-directed mAbCAR T cells enhance survival. Survival of lethally irradiated BALB/c mice that received irradiation alone (black circle), allogeneic C57BL/6 TCD BM (black squares), TCD BM + luc+ A20 alone (white squares in black), TCD BM + luc+ A20 + allogeneic isotype-mAbCAR Tcons (red triangles), TCD BM + luc+ A20 + allogeneic SDF1-mAbCAR Tcons (white circles in blue). One representative of two consecutive experiments is reported; at least 3 mice/group were used. Log-rank survival test; *P < 0.05 for differences between the groups that received luc+ A20 alone and luc+ A20 + allogeneic SDF1-mAbCAR Tcons.

Figure 4. mAbCAR Tregs retain phenotype and function.

(A) mAbCAR Tregs retain FoxP3 expression. Histograms of sample FoxP3 staining (dark blue) in comparison to isotype control (light blue) of FoxP3+ selected Tregs. FoxP3 intranuclear expression in untransfected Tregs, FITC-isotype-mAbCAR Tregs, and FITC-MAdCAM1-mAbCAR Tregs. Mean fluorescence intensity (MFI) of 1 representative of 3 consecutive experiments is reported. (B) mAbCAR Tregs retain STAT5 phosphorylation. Frequency of phosphorylated STAT5 expression in untransfected and transfected Tregs. One representative of two consecutive experiments is reported. (C) mAbCAR Treg TCRβ repertoire is unchanged. The TCRβ CDR3 clone frequency was obtained by TCR nucleotide sequencing. Pearson correlation between untransfected and transfected Tregs is r = 0.92, P < 2.2–16. One representative of two consecutive experiments is reported. (D) Exposure to FITC-coupled antibodies activates mAbCAR Tregs. Whiskers plots represent percentage of expression of selected markers (CD69, LAG3, PD1) before or after in vitro exposure to FITC (24 hours) in mAbCAR Tregs. Data show marker expression in untransfected Tregs (red), mock transfected Tregs (black), and mAbCAR Tregs (gray). n = 2 independent experiments. (E) FITC mAbCAR expression and antibody binding increase Treg proliferation. Percentage of proliferation of untransfected Tregs, FITC-isotype-mAbCAR Tregs, and FITC-MAdCAM1-mAbCAR Tregs after culture with anti-CD3/CD28 beads, measured through cell trace violet dilution. Sample FACS analysis is also shown. One representative of two consecutive experiments is reported. (F) mAbCAR Tregs suppress activated T cells in vitro. Percentage of suppression of T cell proliferation by untransfected Tregs, mAbCAR Tregs, FITC-isotype-mAbCAR Tregs, and FITC-MAdCAM1-mAbCAR Tregs after 4 days stimulation with irradiated allogeneic splenocytes at T cell/Treg ratios of 1:1, 1:2, 1:4, and 1:8, measured through cell trace violet analysis. One representative of two consecutive experiments is reported. (G) mAbCAR Tregs prevent GVHD in vivo. Survival of lethally irradiated BALB/c mice that received allogeneic irradiation alone, TCD BM alone, TCD BM + Tcons, TCD BM + Tcons + untransfected Tregs, TCD BM + Tcons + mAbCAR Tregs, and TCD BM + Tcons + FITC-isotype-mAbCAR Tregs. One representative of three consecutive experiments is reported; at least 3 mice/group were used. Log-rank survival test; for differences between TCD BM + Tcons versus TCD BM + Tcons + untransfected Tregs or TCD BM + Tcons + mAbCAR Tregs or TCD BM + Tcons + FITC-isotype-mAbCAR Tregs. (A, B, and D–F) Two-tailed Student’s t test; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. FITC-H-2Dd-mAbCAR Tregs induce tolerance to allogeneic pancreatic islet grafts if directed against the islet MHC-I alloantigen.

(A) Experimental scheme. Recipient C57BL/6 mice were conditioned with low-dose total body irradiation, followed by allogeneic islet cell transplantation of BALB/c donor islets under the right kidney subcapsule. On the same day of transplant, recipient mice also received sham or mAbCAR Tregs either coated with FITC-conjugated isotype or a mAb against H-2Dd expressed by BALB/c tissue. (B) FITC-H-2Dd-mAbCAR Tregs cause enhanced protection of islet allografts. Fold change over time of BLI uptake (radiance) from mice that received luc+ pancreatic islet graft alone (black circles), luc+ pancreatic islet graft + FITC-isotype-mAbCAR Tregs (blue circles), and luc+ pancreatic islet graft + FITC-H-2Dd-mAbCAR Tregs (red circles). Values of fold change in BLI uptake below 1 have been censored. Data are pooled from 2 consecutive experiments; at least 2 mice per group were used in each experiment. ANOVA test with Bonferroni post-test; mean ± SEM; **P < 0.001 is reported for differences between the group that received luc+ pancreatic islet graft + FITC-H-2Dd-mAbCAR Tregs versus the group that received luc+ pancreatic islet graft alone or luc+ pancreatic islet graft + FITC-isotype-mAbCAR Tregs. (C) BLI reveals in vivo persistence of pancreatic islet allografts after FITC-H-2Dd-mAbCAR Treg transfer. Representative bioluminescent images of luc+ pancreatic islets at 4 weeks after islet transplant. (D) Mice receiving FITC-H-2Dd-mAbCAR Tregs show reduced CD8+ T cell infiltrate in islet allografts. Percentage of pancreatic islet graft infiltration by host-type CD8+ T cells in mice that received no Treg treatment (black), FITC-isotype-mAbCAR Tregs (blue), FITC-H-2Dd-mAbCAR Tregs (red) at 10 days after transplant. Data are representative of 1 of 2 consecutive experiments. Two-tailed Student’s t test; mean ± SEM; *P < 0.05. (E) FITC-H-2Dd-mAbCAR Tregs allow for prolonged survival of pancreatic islet allografts. Percentage of pancreatic islet graft survival at 6 weeks after transplant in mice that received no Treg treatment (black), FITC-isotype-mAbCAR Tregs (blue), and FITC-H-2Dd-mAbCAR Tregs (red). Data are pooled from 2 consecutive experiments.

Figure 6. FITC-H-2Dd-mAbCAR Tregs home and expand in proximity to allogeneic pancreatic islet grafts.

(A) FITC-H-2Dd-mAbCAR Tregs show enhanced localization to the islet allograft. Representative BLI images of sublethally irradiated mice that received a graft of allogeneic pancreatic islets alone, pancreatic islets + luc+ FITC-isotype-mAbCAR Tregs, and pancreatic islets + luc+ FITC-H-2Dd-mAbCAR Tregs at day 10 after adoptive transfer. Data are representative of 1 of 3 consecutive experiments. (B) BLI-based quantification of FITC-H-2Dd-mAbCAR Tregs in proximity of the graft. The graph represents BLI signal from standardized regions of interest in left kidney area (graft site) in sublethally irradiated mice that received a graft of allogeneic pancreatic islets alone (black), pancreatic islets + luc+ FITC-isotype-mAbCAR Tregs (blue), and pancreatic islets + luc+ FITC-H-2Dd-mAbCAR Tregs (red) at day 3, 5, 7 and 10 after adoptive transfer. Data are representative of 1 of 3 consecutive experiments; at least 5 mice per group were used. ANOVA test with Bonferroni post-test; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001. (C) Mice receiving FITC-H-2Dd-mAbCAR Tregs show improved islet allografts. Representative hematoxylin and eosin–stained histologic sections of allogeneic pancreatic grafts in mice that received no Treg treatment, GFP+-FITC-isotype-mAbCAR Tregs, or GFP+-FITC-H-2Dd-mAbCAR Tregs 10 days after transplantation and Treg transfer (arrows = pancreatic islets under the kidney capsule). Original magnification, ×20. (D) FITC-H-2Dd-mAbCAR Treg localization in proximity of the grafts. Confocal microscopy analysis demonstrates presence of transferred Tregs (GFP+, white, red arrows) in proximity of islet grafts (insulin, green) only in mice that received GFP+-FITC-H-2Dd-mAbCAR Tregs. Data are representative of 1 of 3 consecutive experiments; at least 5 mice/group were used. Original magnification, ×63; ×200 (insets). (E) Mice receiving FITC-H-2Dd-mAbCAR Tregs show increased Treg numbers. Percentage of transferred GFP+ Tregs in spleens of mice that received no Treg treatment, FITC-isotype-mAbCAR Tregs, and FITC-H-2Dd-mAbCAR Tregs at 10 days after transplant. Data are representative of 1 of 2 consecutive experiments; at least 4 mice per group were used. (F) Mice receiving FITC-H-2Dd-mAbCAR Tregs show increased Treg activation. Expression of CD25 and CD69 was assessed by flow cytometry in previously transferred GFP+ Tregs reisolated from spleens of mice that received FITC-isotype-mAbCAR Tregs (blue) or FITC-H-2Dd-mAbCAR Tregs (red) at 10 days after transplant. Data are representative of 1 of 2 consecutive experiments; at least 4 mice/group were used.

Figure 7. FITC-H-2Dd-mAbCAR Tregs acquire antigen specificity after in vivo transfer.

(A) Experimental scheme. Mice that were previously transplanted with allogeneic pancreatic islet graft in the left kidney capsule received a secondary double skin graft MHC matched with the previously transplanted pancreatic graft (upper grafts) or “third party” (lower grafts). Data are representative of 2 consecutive experiments; at least 4 mice/group were used. Skins have been also transplanted in inverted position (“third-party” skins as upper grafts and MHC-matched skins as lower graft) in order to avoid technical bias. (B) Mice treated with FITC-H-2Dd-mAbCAR Tregs show alloantigen-specific protection of matched skin grafts. Survival of the skin graft that was MHC matched with the previously transplanted allogeneic islet graft in mice that received no Treg treatment (black squares), or FITC-isotype-mAbCAR Tregs (blue squares), or FITC-H-2Dd-mAbCAR Tregs (red squares). Log-rank survival test; *P < 0.05 is reported for differences between the group that received FITC-H-2Dd-mAbCAR Tregs versus the group that received no Treg treatment or FITC-isotype-mAbCAR Tregs. (C) FITC-H-2Dd-mAbCAR Tregs do not protect “third-party” skin grafts. Survival of the skin graft that was “third-party” with the previously transplanted allogeneic islet graft and the infused Tregs in mice that received no Treg treatment (black squares), or FITC-isotype-mAbCAR Tregs (blue squares), or FITC-H-2Dd-mAbCAR Tregs (red squares). Log-rank survival test; no statistical significance was detected between the three groups.