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. 2022 Sep 8;7(17):e160674.
doi: 10.1172/jci.insight.160674.

Murine CAR19 Tregs suppress acute graft-versus-host disease and maintain graft-versus-tumor responses

Sara Bolivar-Wagers  1 , Michael L Loschi  1 , Sujeong Jin  1 , Govindarajan Thangavelu  1 , Jemma H Larson  1 , Cameron S McDonald-Hyman  2 , Ethan G Aguilar  1 , Asim Saha  1 , Brent H Koehn  1 , Mehrdad Hefazi  3 , Mark J Osborn  1 , Michael C Jensen  4 , John E Wagner  1 , Christopher A Pennell  5 , Bruce R Blazar  1
Affiliations

Murine CAR19 Tregs suppress acute graft-versus-host disease and maintain graft-versus-tumor responses

Sara Bolivar-Wagers et al. JCI Insight. .

"V体育平台登录" Abstract

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) efficacy is complicated by graft-versus-host disease (GVHD), a leading cause of morbidity and mortality VSports手机版. Regulatory T cells (Tregs) have shown efficacy in preventing GVHD. However, high Treg doses are often required, necessitating substantial ex vivo or in vivo expansion that may diminish suppressor function. To enhance in vivo suppressor function, murine Tregs were transduced to express an anti-human CD19 chimeric antigen receptor (hCAR19) and infused into lethally irradiated, hCD19-transgenic recipients for allo-HSCT. Compared with recipients receiving control transduced Tregs, those receiving hCAR19 Tregs had a marked decrease in acute GVHD lethality. Recipient hCD19 B cells and murine hCD19 TBL12-luciferase (TBL12luc) lymphoma cells were both cleared by allogeneic hCAR19 Tregs, which was indicative of graft-versus-tumor (GVT) maintenance and potentiation. Mechanistically, hCAR19 Tregs killed syngeneic hCD19+ but not hCD19- murine TBL12luc cells in vitro in a perforin-dependent, granzyme B-independent manner. Importantly, cyclophosphamide-treated, hCD19-transgenic mice given hCAR19 cytotoxic T lymphocytes without allo-HSCT experienced rapid lethality due to systemic toxicity that has been associated with proinflammatory cytokine release; in contrast, hCAR19 Treg suppressor function enabled avoidance of this severe complication. In conclusion, hCAR19 Tregs are a potentially novel and effective strategy to suppress GVHD without loss of GVT responses. .

Keywords: Bone marrow transplantation; Cancer immunotherapy; Immunology; Stem cell transplantation; Transplantation. V体育安卓版.

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Figures

Figure 1
Figure 1. Generation of hCAR19 Tregs.
(A) Schema of hCAR19 and tEGFR Treg generation. (B) Schematic representation of hCAR19 construct in a pMP71 vector backbone. scFv, single chain variable fragment; VL, variable light chain; VH, variable heavy chain; TM, transmembrane domain; GMCSFRss, GM-CSF receptor α chain signal sequences. (C) Representative histogram plots of truncated human epidermal growth factor receptor (tEGFR) expression in hCAR19 and tEGFR control transduced Tregs prior to experimental use. FMO, fluorescence minus one; pre-E, before tEGFR-positive enrichment; post-E, following tEGFR-positive enrichment. (D) Percentage of tEGFR expression in nontransduced (NT) Tregs, before and after tEGFR enrichment of hCAR19 and tEGFR transduced Tregs. (E) Histogram plots of CD25 and Foxp3 expression in NT, tEGFR, and hCAR19 Tregs. (F) Representative FACS plot of CD25+Foxp3+ transduced hCAR19 Tregs prior to experimental use.
Figure 2
Figure 2. hCAR19 Tregs deplete hCD19 B cells and prevent systemic toxicities in a syngeneic mouse model.
hCD19TGTg/0 recipient mice injected with 300 mg/kg of cyclophosphamide (Cy) a day prior to adoptive cell transfer (ACT) with C57BL/6 hCAR19 or tEGFR Tregs, or hCAR19 or tEGFR CD8+ T cells (CTL). (A) Representative flow cytometry plots and quantification (B) of hCD19+ lymphocytes in the spleen on day 5 after ACT. Cy, n = 4; hCAR19 CD8+ CTLs, n = 3; tEGFR CD8+ CTLs, n = 3; hCAR19 Tregs, n = 4; tEGFR Tregs, n = 3. Data are representative from 2 independent experiments. (C) Survival. (D) Percentage body weight. (E) Clinical scores. Unmanipulated, n = 4; Cy, n = 4; hCAR19 CD8+ CTLs, n = 4; tEGFR CD8+ CTLs, n = 3; hCAR19 Tregs, n = 3; tEGFR Tregs, n = 3. Data are representative of 3 independent experiments. Statistics are shown on day 5 after ACT. Student’s t test with Bonferroni correction for multiple comparison was used for statistical analysis. Log rank test was used to analyze survival curves. Error bars indicate the standard deviation of the mean. *: P < 0.5; **: P < 0.01; ****: P < 0.0001.
Figure 3
Figure 3. hCAR19 Tregs have antitumor responses in the absence of Tcons in a syngeneic tumor model.
Survival (A) and average radiance (B) of hCD19TGTg/0 mice after undergoing a lethal irradiation prior to receiving C57BL/6 bone marrow (BM), BM with hCD19 TBL12luc cells, or BM with hCD19 TBL12luc cells and either tEGFR or hCAR19 Tregs or CTLs. CTLs in this experiment were CD8+ T cells. Survival: BM, n = 10; hCD19 TBL12luc, n = 9; tEGFR Tregs, n = 11; hCAR19 Tregs, n = 9; tEGFR CTLs, n = 11; hCAR19 CTLs, n = 12. Data are pooled from 2 independent experiments. Average radiance: BM, n = 6; hCD19 TBL12luc, n = 6; tEGFR Tregs, n = 7; hCAR19 Tregs, n = 6; tEGFR CTLs, n = 6; hCAR19 CTLs, n = 7. Only 3–4 mice were imaged per group. Data are pooled from 2 independent experiments. (C) Representative images of hCD19TGTg/0 mice on day 7 and 14 after syngeneic HSCT. D7 radiance scale: 10-60 x 106; D14 radiance scale: 0.2-1 x 109. Student’s t test with Bonferroni correction for multiple comparison was used for statistical analysis. Log rank test was used to analyze survival curves. Error bars indicate the standard deviation of the mean. *:<0.5; **: <0.01; ***:<0.001; ****:<0.0001.
Figure 4
Figure 4. hCAR19 Tregs have increased expression of killing markers after antigen-specific activation and engage in in vitro killing of hCD19 TBL12.
(A–F) Frequency of hCAR19 or tEGFR Tregs expressing granzyme A (GzA), perforin (perf), CD107α, and granzyme B (GzB) after 48-hour stimulation in an hCD19 Fc–coated plate. hCAR19 Tregs, n = 4; tEGFR Tregs, n = 4. (G) IncuCyte in vitro killing assay with TBL12 and hCD19 TBL12luc tumor cells stained with Far Red dye and the IncuCyte Caspase-3/7 green apoptosis dye. Effector-to-target (E/T) ratio used was 2:1. n = 3 for all groups. (H) A 48-hour flow cytometry in vitro killing assay using hCD19 TBL12luc with sorted Foxp3+GFP+ Tregs the day of the experiment using a 5:1 E/T ratio in all groups except where noted to be 10:1. n = 6 for all groups. (I) IncuCyte in vitro killing assay using the perforin inhibitor concanamycin A (CMA) and GzB inhibitor Z-AAD-CMK with hCD19 TBL12luc tumor. E/T ratio used was 2:1. n = 5 for all groups, except hCAR19 Treg and hCAR19 Treg GzB had n = 3. (J) A 48-hour flow cytometry in vitro killing assay using hCD19 TBL12luc at an E/T ratio of 5:1. n = 6 for all groups. Statistics for IncuCyte experiments were done at the 48-hour time point. Data in this figure are representative from 2 independent experiments. Student’s t test with Bonferroni correction for multiple comparisons was used for statistical analysis. Unpaired t test (1 tailed) was used for single comparisons. Error bars indicate the standard deviation of the mean. ***:<0.001; ****:<0.0001.
Figure 5
Figure 5. hCAR19 Tregs reduce aGVHD severity and mortality relative to hCAR19 CTLs.
(A) Survival for hCD19TGTg/0 recipient mice after undergoing a lethal irradiation prior to receiving BALB/c BM only; BM with Tcons; or BM with Tcons and hCAR19 Tregs, hCAR19 CD4+ CTLs, or hCAR19 CD8+ CTLs. BM, n = 6; Tcons, n = 6; hCAR19 Tregs, n = 6; hCAR19 CD4+ CTLs, n = 6; hCAR19 CD8+ CTLs, n = 6. Data are representative from 2 independent experiments. (B) Clinical GVHD scores: 0, no disease; 10, severe disease. (C) Clinical scores quantified on day 28. (D) Percentage body weight. Dotted line represents 75% body weight. (E) Percentage body weight quantified on day 28. Student’s t test with Bonferroni correction for multiple comparisons was used for statistical analysis. Log rank test was used to analyze survival curves. Error bars indicate the standard deviation of the mean. *:<0.5; **: <0.01; ***:<0.001; ****:<0.0001.
Figure 6
Figure 6. hCAR19 Tregs reduce aGVHD severity and improve overall survival in an hCD19-dependent manner.
(A) Percentage suppression by NT, tEGFR, or hCAR19 Tregs of CD8+ T cells in the presence of WT or hCD19-containing B cells. (B) Survival for hCD19TGTg/0 recipient mice after undergoing a lethal irradiation prior to receiving 5 × 106 BALB/c BM; BM with 2.5 × 106 Tcons; or BM with Tcons and either 1.25 × 106 hCAR19 or tEGFR Tregs; or WT C57BL/6 recipient mice receiving BALB/c BM with Tcons and hCAR19 Tregs (hCAR19 Treg, WTR). BM, n = 5; Tcons, n = 8; tEGFR Tregs, n = 6; hCAR19 Tregs, n = 6; hCAR19 Treg, WTR, n = 5. Data are representative from 4 independent experiments. (C and D) Clinical GVHD scores: 0, no disease; 10, severe disease. (C) Clinical GVHD scores quantified on day 33. (E) Percentage body weight quantified on day 33. (F) Percentage body weight. Student’s t test with Bonferroni correction for multiple comparisons was used for statistical analysis of weights and clinical scores. Log rank test was used to analyze survival curves. Error bars indicate the standard deviation of the mean. *:<0.5; **: <0.01; ***:<0.001; ****:<0.0001.
Figure 7
Figure 7. hCAR19 Tregs have increased expansion and suppression of activated T cells in the colon following allo-HSCT.
(A) Images of hCD19TGTg/0 mice on day 3 and 5 after receiving BALB/c BM with Tcons and luciferase+ hCAR19 or tEGFR Tregs. Radiance scale of 1.0-4.0 x 105. (B) Average radiance of luciferase+ hCAR19 or tEGFR Tregs from day 3 to 30 after allo-HSCT. tEGFR Tregs, n = 8; hCAR19 Tregs, n = 8. (C) Frequency of CD25+Foxp3+ Tregs in the spleen on day 5 after allo-HSCT. Tcons, n = 4; tEGFR Tregs, n = 4; hCAR19 Tregs, n = 3. (D–J) Colon was harvested on day 14 after allo-HSCT in hCD19TGTg/0 recipient mice that received BALB/c BM with Tcons, or BM with Tcons and tEGFR or hCAR19 Tregs. (E and F) Treg to CD4+ and CD8+ T cell ratios. (G–J) Absolute number of TNF-α+ and IFN-γ+ CD4+ and CD8+ T cells. Tcons, n = 4; tEGFR Tregs, n = 4; hCAR19 Tregs, n = 4. SPL, spleen; LI, large intestine. Data from all experiments are representative from 2 independent experiments. Student’s t test with Bonferroni correction for multiple comparison was used for statistical analysis. Error bars indicate the standard deviation of the mean. *:<0.5; **: <0.01; ***:<0.001; ****:<0.0001.
Figure 8
Figure 8. hCAR19 Tregs maintain GVT responses.
(A) Absolute number and frequency of hCD19+ and mouse CD19+ (mCD19+) (B) lymphocytes in the spleen on day 5 of hCD19TGTg/0 recipient mice after undergoing a lethal irradiation prior to receiving BALB/c BM with Tcons or BM with Tcons and either hCAR19 or tEGFR Tregs. Data are pooled from 2 independent experiments. Tcons, n = 8; tEGFR Tregs, n = 7; hCAR19 Tregs, n = 6. (C) Representative flow plots demonstrating hCD19+ lymphocyte depletion in hCAR19 Treg–treated mice on day 5 after allo-HSCT. hCD19 lymphocytes were gated from live B220 cells. (D) Survival of hCD19TGTg/0 recipient mice after undergoing a lethal irradiation prior to receiving BALB/c BM, BM with hCD19 TBL12, BM with hCD19 TBL12 with Tcons, or BM with hCD19 TBL12 and Tcons and either hCAR19 or tEGFR Tregs. Data are representative from 2 independent experiments. BM, n = 7; hCD19 TBL12luc, n = 9; Tcons, n = 8; tEGFR Tregs, n = 8; hCAR19 Tregs, n = 9. (E) Average radiance of hCD19 TBL12luc. Average radiance: BM, n = 4; hCD19 TBL12luc, n = 4; tEGFR Tregs, n = 4; hCAR19 Tregs, n = 4. Only 3–5 mice were imaged per group. Data are representative from 2 independent experiments. (F) Images from day 14 and 21 after allo-HSCT. Student’s t test with Bonferroni correction for multiple comparisons was used for statistical analysis. Log rank test was used to analyze survival curves. Error bars indicate the standard deviation of the mean. *:<0.5; **: <0.01; ****:<0.0001.
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