Methods and kits for reducing the risk of allograft rejection

Abstract

Methods of reducing allograft rejection or the risk of allograft rejection in a subject are described. The methods include metabolically labelling an allograft tissue or organ with a chemical tag (such as dinitrophenyl), transplanting the labelled tissue or organ, and administering to the transplant recipient T regulatory (Treg) cells expressing a chimeric antigen receptor (CAR) specific for the chemical tag. The CAR-expressing Treg cells are stimulated upon binding to the chemical tag displayed on the transplanted tissue or organ, leading to activation of the Treg cells and suppression of effector CD8+ T cells, thereby generating of an immunosuppressive tissue environment that reduces the risk of allograft rejection. Kits that include a chemical tag, engineered CAR Treg cells specific for the chemical tag, and / or a viral vector encoding a CAR specific for the chemical tag are also described.

Description

[0001]7950-111351-02METHODS AND KITS FOR REDUCING THE RISK OF ALLOGRAFT REJECTION CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63 / 550,246, filed February 6, 2024, which is herein incorporated by reference in its entirety. FIELD This disclosure concerns methods and kits for decreasing the risk of allograft rejection in a subject by metabolically labeling the allograft with a chemical tag and administering to the subject T regulatory (Treg) cells expressing a chimeric antigen receptor (CAR) specific for the chemical tag. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT This invention was made with government support under R01CA274738 and R21CA270872 awarded by the National Institutes of Health and 2143673 awarded by the National Science Foundation. The government has certain rights in the invention. INCORPORATION OF ELECTRONIC SEQUENCE LISTING The electronic sequence listing, submitted herewith as an XML file named 7950- 111351-02.xml (11,641 bytes), created on January 23, 2025, is herein incorporated by reference in its entirety. BACKGROUND Transplantation has been widely explored for the replacement of diseased cells, tissues, and organs. However, immune rejection, as a result of the attack of host immune cells towards transplanted cells, remains a major hurdle for transplant survival (Iske et al., Cell Mol Immunol 16:343-349, 2019; LaRosa et al., J Immunol 178:7503-7509, 2007). Current practice involves the long-term administration of immunosuppressive drugs such as rapamycin, tacrolimus, and mycophenolate to suppress the activity of T cells, antigen- presenting cells, and other effector immune cells (Saunders et al., Kidney Int 59:3-16, 2001; Webster et al., Transplantation 81:1234-1248, 2006; Grinyo and Cruzado, Am J Transplant 6:1991-1999, 2006). However, these broad-spectrum drugs inevitably impair the immune system and cause susceptibility to infections and complications (Meier‐Kriesche et al., Am J Transplant 5:2273-2280, 2005; Kang and Park, Curr Opin Rheumatol 15:528-534, 2003;7950-111351-02Adams et al., J Hepatol 62:S170-S185, 2015). Extensive efforts have also been made to improve the specificity of immune inhibition, by developing immunosuppressive agents capable of specifically inhibiting transplant-attacking immune cells. Among these efforts, regulatory T (Treg) cells that can suppress effector T cells via inhibitory cytokines, cytolysis, or metabolic disruption and play a crucial role in maintaining peripheral tolerance, have been a target of interest (Wood and Sakaguchi, Nat Rev Immunol 3:199-210, 2003; Romano et al., Front Immunol 10:43, 2019). For example, allograft-specific Tregs were enriched via in vitro allogeneic stimulation and adoptively transferred for transplant protection (Theil et al., Cytotherapy 17:473-486, 2015; Putnam et al., Am J Transplant 13:3010-3020, 2013; Peters et al., PloS One 3:e2233, 2008). To further improve the specificity, chimeric antigen receptor (CAR) Tregs specific to transplant-associated antigens have also been actively explored (Ferreira et al., Nat Rev Drug Discov 18:749-769, 2019; MacDonald et al., J Clin Invest 126:1413-1424, 2016; Dawson et al., Sci Transl Med 12:eaaz3866, 2020). Human leukocyte antigen class I molecule A*02 (HLA-A*02) is an allogenic antigen commonly utilized for the design of transplant-specific CAR Tregs (Noyan et al., Am J Transplant 17:917-930, 2017; Proics et al., Gene Ther 30:309-322, 2023; Schreeb et al., Kidney Int Rep 7:1258-1267, 2022), but is only applicable to the scenario when transplantation from HLA- A*02-positive donors to HLA-A*02-negative recipients occurs. Other than HLA-A*02, attempts to identify endogenous transplant-specific antigens have been largely unsuccessful. SUMMARY Provided herein are methods of reducing allograft rejection or the risk of allograft rejection in a subject. In some aspects, the method includes metabolically labeling an allograft tissue or organ with a unique chemical tag; transplanting the labelled allograft tissue or organ in the subject; and administering to the subject a therapeutically effective amount of T regulatory (Treg) cells expressing a chimeric antigen receptor (CAR) that specifically binds the chemical tag displayed on the allograft tissue or organ. The chemical tag is not present in the transplant recipient and thus serves as a transplant-specific antigen for targeting with a CAR. The CAR-expressing Treg cells are stimulated upon binding to the chemical tag displayed on the transplanted tissue or organ, leading to activation of the Treg cells and suppression of effector CD8+ T cells and dendritic cells, thereby generating of an immunosuppressive tissue environment that reduces the risk of allograft rejection. In some aspects of the disclosed methods, the chemical tag includes dinitrophenyl (DNP). In some examples, metabolic labelling of the allograft tissue or organ includes7950-111351-02contacting the allograft tissue or organ with a DNP sugar, such as but not limited to N-(2,4- dinitrophenylamino)-acetyl mannosamine (ManDNP-2), tetraacetyl-N-(2,4- dinitrophenylamino) acetyl mannosamine (Ac4ManDNP-2), N-(2,4-dinitrophenylamino)- caprylic mannosamine (ManDNP-6), tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic mannosamine (Ac4ManDNP-6), N-(2,4-dinitrophenylamino)-acetyl galactosamine (GalDNP- 2), tetraacetyl-N-(2,4-dinitrophenylamino) acetyl galactosamine (Ac4Gal-DNP-2), N-(2,4- dinitrophenylamino)-caprylic galactosamine (GalDNP-6), tetraacetyl-N-(2,4- dinitrophenylamino)-caprylic galactosamine (Ac4Gal-DNP-6), N-(2,4-dinitrophenylamino)- acetyl neuraminic acid (Neu5DNP-2), tetraacetyl-N-(2,4-dinitrophenylamino)-acetyl neuraminic acid (Ac4Neu5DNP-2), N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Neu5DNP-6), or tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Ac4Neu5DNP-6). The Treg cells can be, for example, autologous Treg cells isolated from the subject, allogeneic Treg cells obtained from a donor, or Treg cells produced from universal stem cells. Also provided herein are kits that include a chemical tag and / or a viral vector encoding a chimeric antigen receptor (CAR) that specifically binds the chemical tag. In some aspects, the chemical tag includes DNP or a DNP analog, fluorescein or a fluorescein derivative, biotin or a biotin derivative, sulfanilamide or a sulfanilamide derivative, a nitrated amino acid, a modified nucleotide or nucleoside, a biocompatible polymer, or an oxidized lipid. In some examples, the chemical tag includes DNP, such as a DNP sugar. The kit can optionally include other components to carry out metabolic labelling of the allograft tissue or organ with the chemical tag, transduction of Treg cells with the viral vector and / or administering the transduced Treg cells to a subject. The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS FIGS.1A-1C. Schematics of a universal approach to developing transplant-specific CAR Tregs. (FIG.1A) Transplants are metabolically labeled with chemical tags (e.g., dinitrophenyl (DNP) group) via the metabolic glycoengineering processes of unnatural sugars. (FIG.1B) Anti-DNP CAR Tregs recognize and become stimulated by DNP-labeled transplants, leading to suppression of CD8+T cells and dendritic cells in a DNP-specific7950-111351-02manner. (FIG.1C) In a murine skin allotransplantation model, anti-DNP CAR Tregs are stimulated at the transplant site for the immune protection of skin allografts. FIGS.2A-2L. DNP-sugars metabolically label cells with DNP groups. (FIG.2A) Chemical structure of four DNP-sugars, ManDNP-6, Ac4ManDNP-6, ManDNP-2, and Ac4ManDNP-2. (FIGS. 2B-2L) Cells were incubated with DNP-sugars for three days and cell-surface DNP groups were detected by AF647-conjugated anti-DNP. (FIGS.2B-2E) Mean AF647 fluorescence intensity (FI) of ID8-Cos ovarian cancer cells pretreated with different concentrations of (FIG.2B) ManDNP-6, (FIG.2C) Ac4ManDNP-6, (FIG.2D) ManDNP-2, and (FIG.2E) Ac4ManDNP-2. (FIGS.2F-2G) Mean AF647 FI of primary human keratinocytes (FIG.2F) and CHO cells (FIG.2G) pretreated with 50 µM ManDNP-6, Ac4ManDNP-6, ManDNP-2, and Ac4ManDNP-2. (FIGS.2H-2L) Mean AF647 FI of (FIG. 2H) HEK293 cells, (FIG.2I) LS174T cells, (FIG.2J) A549 cells, (FIG.2K) EL-4 cells, and (FIG.2L) MSCs pretreated with different concentrations of ManDNP-6 for three days. All numerical data are presented as mean ± SD (one-way ANOVA with post hoc Fisher’s LSD test or two-tailed Welch’s t-test was used; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). FIGS.3A-3J. Development of anti-DNP CAR Tregs that can specifically and sensitively recognize DNP. (FIG.3A) The designed CAR construct consists of a hemagglutinin (HA) tag, an extracellular anti-DNP scFv, a CD8 hinge region, a CD28 transmembrane domain, and CD28 and CD3ξ intracellular signaling domains. (FIGS.3B- 3C) CD4+CD25+Tregs isolated from C57BL / 6 mice were transduced with the CAR constructs. After 7 days, transduced cells were purified using the puromycin selection method and analyzed by flow cytometry. (FIG.3B) Representative HA histogram and (FIG. 3C) percentages of HA+Tregs. Un-transfected Tregs were used as controls. (FIGS.3D-3F) Anti-DNP CAR Tregs were cocultured with DNP-OVA for 24 h, and the bounded DNP- OVA was detected with AF647-conjugated anti-DNP. (FIG.3D) Schematic illustration of the study. (FIG.3E) Representative anti-DNP histograms of CAR Tregs and anti-CD19 CAR Tregs after co-incubation with 20 μg / mL DNP-OVA for 24 h. (FIG.3F) Percentage of DNP- OVA+Tregs after co-incubation with different concentrations of DNP-OVA for 24 h. (FIGS. 3G-3H) Representative Helios-DNP flow cytometry plots of (FIG.3G) anti-DNP CAR Tregs and (FIG.3H) anti-CD19 CAR Tregs after incubation with DNP-OVA for 24 h. (FIG.3I) Percentages of DNP+Helios+Tregs after incubation with different concentrations of DNP- OVA for 24 h. (FIG.3J) Percentages of Helios+cells among DNP+anti-DNP CAR Tregs7950-111351-02after incubation with different concentrations of DNP-OVA for 24 h. All numerical data are presented as mean ± SD (one-way ANOVA with post hoc Fisher’s LSD test or two-tailed Welch’s t-test was used; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). FIGS.4A-4H. Anti-DNP CAR Tregs can be activated by DNP-labeled cells and inhibit CD8+T cells and dendritic cells. (FIGS.4A-4C) CHO cells were pre-treated with ManDNP-6 for 3 days and then co-incubated with anti-DNP CAR Tregs for 24 h. (FIG.4A) Schematic illustration of the co-culture study. (FIG.4B) Representative Helios plots and (FIG.4C) percentages of Helios+CAR Tregs after incubation with unlabeled or DNP-labeled CHO cells for 24 h. (FIGS.4D-4E) In vitro CD8+T cell suppression assay. Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD8+T cells were cocultured with CAR Tregs, with or without the presence of DNP-labeled CHO cells, for 3 days. (FIG.4D) Representative CFSE histograms of CD8+T cells for different groups. (FIG.4E) Proliferation index of CD8+ T cells for different groups. (FIG.4F) % of lysed E.G7-OVA by OT-1 cells in the presence of anti-DNP CAR Tregs or anti-CD19 CAR Tregs. E.G7-OVA cells were pretreated with ManDNO-6 or PBS for three days and stained with Calcein AM, followed by the incubation with the mixture of Tregs and OT-1 cells for 24 h. (FIGS.4G- 4H) In vitro dendritic cell (DC) suppression assay. DCs were cocultured with CAR Tregs and DNP-labeled CHO cells for 4 days. (FIG.4G) Representative CD86-CD11c plots of DCs for different groups. (FIG.4H) Percentages of CD86+DCs after different treatments. All numerical data are presented as mean ± SD (one-way ANOVA with post hoc Fisher’s LSD test or two-tailed Welch’s t-test was used; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). FIGS.5A-5J. Anti-DNP CAR Tregs are stimulated by ManDNP-6 loaded skin allografts in vivo and improve the survival of skin allografts. (FIG.5A) Timeframe of study. Back skin (1.0 cm × 1.5 cm) was isolated from Balb / c mice, injected with ManDNP-6 or PBS, and transplanted to C57BL / 6 mice on day 0. Anti-DNP CAR Tregs were intravenously injected on days 2, 3, and 4. On Day 7, skin allografts were harvested for immune cell analysis. (FIG.5B) Representative CD25-HA plots of leukocytes isolated from skin allografts. CD4+cells were pre-gated. (FIG.5C) Percentages of CD25+HA+CAR Tregs among CD4+T cells in the skin allografts. (FIG.5D) Percentages of HA+CAR Tregs among CD4+CD25+Tregs in the skin allografts. (FIG.5E) Representative CD4-CD8 plots of leukocytes isolated from skin allografts. (FIG.5F) Percentages of CD4+or CD8+T cells in the skin allografts. (FIG.5G) CD4+ / CD8+T cell number ratio in skin allografts. (FIG.5H) Timeframe of the skin graft survival study. Back skin (1.0 cm × 1.5 cm) was isolated from7950-111351-02Balb / c mice, injected with ManDNP-6 or PBS, and transplanted to C57BL / 6 mice on day 0. Anti-DNP CAR Tregs (5.0 × 105) were intravenously injected on days 2, 3, and 4. (FIG.5I) Images of skin grafts over time for different groups. (FIG.5J) Survival curves of skin allografts. Graft rejection was defined as the loss of over 25% of transplanted skin. All numerical data are presented as mean ± SD (one-way ANOVA with post hoc Fisher’s LSD test or two-tailed Welch’s t-test was used; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). FIGS.6A-6E. (FIG.6A) Synthetic routes of ManDNP-2 and Ac4ManDNP-2. (FIG. 6B)1H NMR and (FIG.6C)13C NMR spectra of ManDNP-2. (FIG.6D)1H NMR and (FIG. 6E)13C NMR spectra of Ac4ManDNP-2. FIGS.7A-7E. (FIG.7A) Synthetic routes of ManDNP-6 and Ac4ManDNP-6. (FIG. 7B)1H NMR and (FIG.7C)13C NMR spectra of ManDNP-6. (FIG.7D)1H NMR and (FIG. 7E)13C NMR spectra of Ac4ManDNP-6. FIGS.8A-8F. Metabolic labeling of cells with DNP groups. Cells were incubated with DNP-sugars for three days and cell-surface DNP groups were detected by AF647- conjugated anti-DNP. (FIGS.8A-8C) Mean AF647 FI of B16F10 cancer cells pretreated with different concentrations of (FIG.8A) ManDNP-6, (FIG.8B) Ac4ManDNP-6, and (FIG. 8C) ManDNP-2. (FIGS.8D-8G) Mean AF647 FI of ID8 cancer cells pretreated with different concentrations of (FIG.8D) ManDNP-6, (FIG.8E) Ac4ManDNP-6, and (FIG.8F) ManDNP-2. All numerical data are presented as mean ± SD (one-way ANOVA with post hoc Fisher’s LSD test or two-tailed Welch’s t-test was used; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). FIGS.9A-9I. ManDNP-6 can metabolically label various types of cancerous and non-cancerous cells with DNP groups. Cells were incubated with DNP-sugars for three days and cell-surface DNP groups were detected by AF647-conjugated anti-DNP. (FIGS.9A-9B) Percentages of DNP+(FIG.9A) human primary keratinocytes and (FIG.9B) CHO cells after 72-h incubation with 50 µM ManDNP-2, Ac4ManDNP-2, ManDNP-6, and Ac4ManDNP-6. (FIGS.9C-9G) Percentages of DNP+(FIG.9C) HEK293 cells, (FIG.9D) LS174T cells, (FIG.9E) A549 cells, (FIG.9F) EL4 cells, and (FIG.9G) MSC cells after 72-h treatment with different concentrations of ManDNP-6. (FIG.9H) Percentages of DNP+CHO cells after 72-h incubation with ManDNP-6 (ManDNP-6 was replenished every 24 h). (FIG.9I) Percentages of DNP+CHO cells after 72-h incubation with ManDNP-6, with the pre- starvation of cells for 0, 12 or 24 h. All numerical data are presented as mean ± SD (one-way7950-111351-02ANOVA with post hoc Fisher’s LSD test was used; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). FIGS.10A-10G. Development of anti-DNP CAR and anti-CD19 CAR. (FIG.10A) Design of anti-DNP CAR construct consisting of an HA tag, an extracellular anti-DNP scFv, CD8 transmembrane domain, and CD28 and CD3ξ intracellular signaling domains. (FIG. 10B) Design of control anti-CD19 CAR construct consisting of an HA tag, an extracellular anti-CD19 scFv, CD8 transmembrane domain, and CD28 and CD3ξ intracellular signaling domains. (FIGS.10C-10E) Cloning of anti-DNP CAR constructs in pJLM1 vector for lentiviral packaging. The pJLM1 vector (Addgene #91980) was digested at NheI + EcoRI sites, and clonal DNA for FIG.10A) and FIG.10B) were ordered from Twist Bioscience and PCR amplified. The digested vector and PCR fragments were assembled using Gibson assembly and confirmed by whole plasmid sequencing from Plasmidsaurus. (FIG.10C) Agarose gel for (1) PCR amplified anti-DNP CAR construct, (2) digested pLJM1 vector, and (3) PCR amplified control anti-CD19 CAR construct. (FIG.10D) Reconstruct transfer plasmid for anti-DNP CAR. (FIG.10E) Percentages of HA+Tregs after transducing Tregs with different amount of virus titers. CD4+CD25+Tregs isolated from C57BL / 6 mice were transduced with the anti-CD19 CAR construct. After 7 days, transduced cells were purified using the puromycin selection method and analyzed by flow cytometry. FIGS.11A-11C. Anti-DNP CAR Tregs can specifically recognize and become stimulated by DNP-OVA. Anti-DNP CAR Tregs or anti-CD19 CAR Tregs were incubated with different concentrations of DNP-OVA, followed by staining with fluorophore- conjugated anti-CD25, anti-CD4, anti-Helios, and anti-DNP. (FIG.11A) Representative Helios-CD25 FACS plots of CAR Tregs after co-incubation with 20 μg / ml DNP-OVA for 24 h. (FIG.11B) Percentages of CD25+Helios+cells among CD4+cells after incubating CAR Tregs with different concentrations of DNP-OVA for 24 h. (FIG.11C) Representative Helios-DNP flow plots of anti-DNP CAR Tregs and anti-CD19 CAR Tregs after incubation with different concentrations of DNP-OVA for 24 h. FIGS.12A-12B. Anti-DNP CAR Tregs incubated with DNP-OVA exhibit upregulated CD69 expression and altered cytokine secretion profile. (FIG.12A) Percentages of CD69+CAR Tregs after 24-h incubation with different concentrations of DNP-OVA. (FIG.12B) Cytokine secretion profiles of anti-DNP CAR Tregs after 24-h incubation with PBS or DNP-OVA. FIGS.13A-13E. Anti-DNP CAR Tregs are stimulated by DNP-labeled CHO cells. CHO cells were pre-treated with ManDNP-6 for 3 days and then co-incubated with anti-DNP7950-111351-02CAR Tregs or anti-CD19 CAR Tregs for 24 h. (FIG.13A) Representative CD69 histograms and (FIG.13B) percentages of CD69+ Tregs after 24-h incubation with DNP-labeled or unlabeled CHO cells. (FIG.13C) Representative CTLA-4 histograms and (FIG.13D) percentages of CTLA-4+ Tregs after 24-h incubation with DNP-labeled or unlabeled CHO cells. (FIG.13E) Cytokine secretion profiles of anti-DNP CAR Tregs after 24-h incubation with PBS or DNP-labeled CHO cells. FIGS.14A-14B. Anti-DNP CAR Tregs can suppress DCs in a DNP-dependent manner. DCs were cocultured with CAR Tregs and DNP-labeled CHO cells for 4 days. The ratio of anti-DNP CAR Tregs or anti-CD19 CAR Tregs to DCs was set at 1:1 or 5:1. (FIG. 14A) Representative CD80-CD11c plots of DCs for different groups. (FIG.14B) Percentages of CD80+DCs after different treatments. All numerical data are presented as mean ± SD (one-way ANOVA with post hoc Fisher’s LSD test or two-tailed Welch’s t-test was used; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). FIGS.15A-15C. Hematoxylin and eosin (H&E) staining of skin allografts isolated from C57BL / 6 mice receiving (FIG.15A) PBS-treated skin graft only (untreated), (FIG.15B) PBS-treated skin graft + anti-DNP CAR Tregs, and (FIG.15C) ManDNP-6-treated skin graft + anti-DNP CAR Tregs. Scale bars, 500 μm (10× H&E), 250 μm (40× H&E). Back skin (1.0 cm × 1.5 cm) was isolated from Balb / c mice, injected with ManDNP-6 or PBS, and transplanted to C57BL / 6 mice on day 0. Anti-DNP CAR Tregs (5.0 × 105) were intravenously injected on days 2, 3, and 4. Skin allografts were harvested on day 9, sectioned, and stained with H&E. FIGS.16A-16H. Chemical structures of exemplary chemical tags for use with the methods disclosed herein. Exemplary chemical tags include (FIG.16A) DNP and DNP analogs, (FIG.16B) fluorescein and fluorescein derivatives, (FIG.16C) biotin and biotin derivatives, (FIG.16D) sulfanilamide and derivatives, (FIG.16E) nitrated amino acids, (FIG. 16F) modified nucleotides or nucleosides, (FIG.16G) biocompatible polymers (e.g., PEG or OEG oligomers), and (FIG.16H) oxidized lipids. FIG.17. Map of the DNP-targeted CAR construct. SEQUENCES The nucleic acid and amino acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases and amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.7950-111351-02SEQ ID NO: 1 is the nucleotide sequence of an exemplary DNP-targeted CAR construct. SEQ ID NO: 2 is the amino acid sequence of an exemplary DNP-targeted CAR. SEQ ID NO: 3 is the amino acid sequence of a linker peptide. SEQ ID NO: 4 is the amino acid sequence of DNP VL-(G4S)3 Linker-DNP VH-CD8 Hinge-CD28-CD3ζ. SEQ ID NO: 5 is the amino acid sequence of a DNP-specific scFv. SEQ ID NO: 6 is the amino acid sequence of a CD8 hinge region. SEQ ID NO: 7 is the amino acid sequence of a CD28 transmembrane (TM) and signaling domain. SEQ ID NO: 8 is the amino acid sequence of a CD3ζ signaling domain. SEQ ID NO: 9 is the amino acid sequence of an ovalbumin peptide. DETAILED DESCRIPTION I. Abbreviations Ac4Gal-DNP-2 tetraacetyl-N-(2,4-dinitrophenylamino) acetyl galactosamine Ac4Gal-DNP-6 tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic galactosamine Ac4ManDNP-2 tetraacetyl-N-(2,4-dinitrophenylamino) acetyl mannosamine Ac4ManDNP-6 tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic mannosamine Ac4Neu5DNP-2 tetraacetyl-N-(2,4-dinitrophenylamino)-acetyl neuraminic acid Ac4Neu5DNP-6 tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic neuraminic acid BrdU 5-bromo-2'-deoxyuridine CAR chimeric antigen receptor CFSE carboxyfluorescein succinimidyl ester DC dendritic cell DNCB dinitrochlorobenzene DNP dinitrophenyl GalDNP-2 N-(2,4-dinitrophenylamino)-acetyl galactosamine GalDNP-6 N-(2,4-dinitrophenylamino)-caprylic galactosamine H&E hematoxylin and eosin HA hemagglutinin HLA human leukocyte antigen ManDNP-2 N-(2,4-dinitrophenylamino)-acetyl mannosamine7950-111351-02ManDNP-6 N-(2,4-dinitrophenylamino)-caprylic mannosamine Neu5DNP-2 N-(2,4-dinitrophenylamino)-acetyl neuraminic acid Neu5DNP-6 N-(2,4-dinitrophenylamino)-caprylic neuraminic acid NHPA 3-nitro-4-hydroxyphenylacetate NMR nuclear magnetic resonance OEG oligo(ethylene glycol) OVA ovalbumin PEG polyethylene glycol scFv single chain variable fragment TM transmembrane Treg T regulatory VH variable heavy VL variable light II. Summary of Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided: Administration: To provide or give a subject an agent, such as a CAR-expressing T regulatory cell, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal,7950-111351-02intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Allograft: A transplant of an organ, tissue, bodily fluid or cell from one individual to a genetically non-identical individual of the same species. In some aspects herein, the allograft includes skin, blood vessel, bone, bone marrow, stem cells, cartilage, tendon, ligament, nerves, cornea, heart valve, heart, intestine, stomach, kidney, liver, lung, pancreas, or uterus. Allograft rejection: A partial or complete immune response to a transplanted cell, tissue, organ, or the like on or in a recipient of the transplant due to an immune response to the allograft. Allografts can be rejected through either a cell-mediated or humoral immune reaction of the recipient against histocompatibility antigens present on the donor cells. Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen (such as a unique chemical tag, e.g., DNP). Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region, respectively. Together, the VHregion and the VLregion are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians. Antibody variable regions contain "framework" regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; “Paratome CDRs”)7950-111351-02and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online. In addition, the AbRSA tool can be used to determine the CDR boundaries according to Kabat, IMGT or Chothia (online at aligncdr.labshare.cn / aligncdr / abrsa.php). A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VHdomain antibodies, VNARantibodies, camelid VHH antibodies, and VLdomain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains. A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies can be produced by methods known to those of skill in the art. Monoclonal antibodies include humanized monoclonal antibodies. A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species. A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one aspect, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Chemical tag: In the context of the present disclosure, a chemical tag is a unique molecular tag that functions as an antigen for targeting by a CAR. The chemical tag is not present in a transplant recipient and thus serves as a transplant-specific antigen for targeting7950-111351-02with a CAR. The chemical tag is generally small enough to be metabolized, but large enough to elicit the production of antibodies specific for the chemical tag. In some aspects herein, the chemical tag includes dinitrophenyl (DNP) or a DNP analog, fluorescein or a fluorescein derivative, biotin or a biotin derivative, sulfanilamide or a sulfanilamide derivative, a nitrated amino acid, a modified nucleotide or nucleoside, a biocompatible polymer, or an oxidized lipid (see FIGS.16A-16H). Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen- binding portion (such as a scFv or a single-domain antibody) and a signaling domain, such as a signaling domain from a T cell receptor (for example, CD3ζ). Typically, CARs are comprised of an antigen-binding moiety, a hinge region, a transmembrane domain and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ or FcεRIγ. In some instances, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27 and / or DAP10. Contacting: Placement in direct physical association; includes both in solid and liquid form. Linker: In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond a variable heavy chain (or VH domain) to the variable light chain (or VL domain). The terms “conjugating,” “joining,” “bonding” or “linking” refer to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a radionuclide or other molecule to a polypeptide, such as an antibody. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule. Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals. In some examples, the subject is a transplant recipient (for example a subject that has received an organ transplant, such as a liver, heart, lung, or kidney transplant), or a candidate for a transplant recipient. T regulatory (Treg) cell: A subpopulation of T cells that module the immune system, maintain peripheral tolerance, prevent autoimmune disease, and limit chronic inflammatory diseases. However, these cells can also limit beneficial responses by7950-111351-02suppressing sterilizing immunity and limiting anti-tumor immunity. Treg cells are known to downregulate the induction and proliferation of effector T cells, and are thus considered immunosuppressive. Tregs are most commonly identified as CD3+CD4+CD25+FoxP3+cells in both mice and humans. Additional cell surface markers include CD39, CD73, CTLA-4, GITR, LAG-3, LRRC32, and neuropilin-1. Treg cells can also be identified based on the secretion of immunosuppressive cytokines, including TGF-β, IL-10, and IL-35. Methods of isolating Treg cells are known (see, e.g., Gu et al., STAR Protoc 3(4):101740, 2022; Mandapathil et al., J Immunol Methods 346(1-2):55-63, 2009) and numerous kits are commercially available. Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to reduce the risk of allograft rejection in a subject. In one aspect, a therapeutically effective amount of Treg cells expressing a chemical tag-specific CAR, is the amount necessary to reduce allograft rejection by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, compared to allograft rejection in the absence of treatment. Transplant: Graft of an organ, tissue or cells from one subject to another subject. Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. In some aspects, the vector is a virus vector, such as a lentivirus, adeno-associated virus, or adenovirus vector. III. Methods and Kits for Reducing Risk of Allograft Rejection Considering the challenges in identifying endogenous transplant-specific antigens, the present disclosure contemplates introducing artificial antigens onto transplanted cells to provide a method for developing universal transplant-specific CAR Tregs. Disclosed herein is a facile approach to introduce unique chemical tags (e.g., dinitrophenol (DNP)) onto transplanted cells via metabolic glycoengineering processes of unnatural sugars (FIG.1A) (Xie et al., Proc Natl Acad Sci USA 113:5173-5178, 2016; Wang et al., Nature Mater 19:1244-1252, 2020; Chang et al., J Am Chem Soc 132:9516-9518, 2010; Wang and Mooney, Nat Chem 12:1102-1114, 2020; Han et al., Nat Commun 14:5049, 2023; Laughlin and Bertozzi, Nat Protoc 2:2930-2944, 2007), and develop CAR Tregs that can specifically7950-111351-02recognize the introduced artificial chemical tags (FIG.1B). These chemical tags are not present in the recipients and thus serve as transplant-specific antigens for CAR Treg design. Among the various types of chemical tags that can be introduced via metabolic glycan labeling (Prescher et al., Nature 430:873-877, 2004; Bo et al., Proc Natl Acad Sci USA 120(36):e2302342120, 2023; Fan et al., Nat Chem Biol 18:625-633, 2022; Wang, H. et al., Nat Chem Biol 13:415-424, 2017; Wang et al., Angew Chem Int Ed Engl 55:5452-5456, 2016; Bo et al., Biomaterials, 238:119843, 2020), DNP with a relatively small size and availability of known antibody sequences was used for the studies disclosed herein (Parker et al., J Am Chem Soc 131:16392-16394, 2009; McEnaney et al., ACS Chem Biol 7:1139-1151, 2012). The data show that various types of cancer cells and non-cancerous cells including human primary keratinocytes and mouse mesenchymal stromal cells can be metabolically labeled with DNP groups via the metabolic glycoengineering processes of DNP-sugars. The DNP-labeled cells induce the activation and proliferation of anti-DNP CAR Tregs in vitro and in vivo. The anti-DNP CAR Tregs suppress effector T cells and dendritic cells in a DNP- specific manner, and prolong the survival of skin transplants in a murine skin allotransplantation model (FIG.1C). Methods of reducing allograft rejection or the risk of allograft rejection in subject are described. In some aspects, the method includes metabolically labelling an allograft tissue or organ with a chemical tag, transplanting the labelled tissue or organ in the subject, and administering to the transplant recipient T regulatory cells expressing a CAR specific for the chemical tag. The CAR-expressing Treg cells are stimulated upon binding to the chemical tag displayed on the transplanted tissue or organ, leading to activation of the Treg cells and suppression of effector CD8+ T cells, thereby generating of an immunosuppressive tissue environment that reduces the risk of allograft rejection. The chemical tag can be any suitable molecule that is small enough to be metabolized, but large enough to induce the generation of chemical tag-specific antibodies. In some aspects, the chemical tag is dinitrophenyl (DNP) or a DNP analog (FIG.16A), fluorescein or a fluorescein derivative (FIG.16B), biotin or a biotin derivative (FIG.16C), sulfanilamide or a sulfanilamide derivative (FIG.16D), a nitrated amino acid (FIG.16E), a modified nucleotide or nucleoside (FIG.16F), a biocompatible polymer (FIG.16G), or an oxidized lipid (FIG.16H). In some examples in which the chemical tag is a DNP analog, the chemical tag includes dinitrochlorobenzene (DNCB), 3-nitro-4-hydroxyphenylacetate (NHPA), 2,4- dinitrophenol, picric acid, 2,5-dinitrophenol, m-nitrophenol, 2,6-dinotrophenol, or p- nitroaniline. In some examples in which the chemical tag is a nitrated amino acid, the7950-111351-02chemical tag includes nitrotyrosine. In some examples in which the chemical tag is a modified nucleotide or nucleoside, the chemical tag includes 5-bromo-2'-deoxyuridine (BrdU) or 5-methylcytidine. In some examples in which the chemical tag is a biocompatible polymer, the chemical tag includes polyethylene glycol (PEG) or oligo(ethylene glycol) (OEG). In yet other examples in which the chemical tag is an oxidized lipid, the chemical tag includes 4-hydroxynonenal. In particular aspects, the chemical tag includes DNP. In some examples, metabolically labeling the allograft tissue or organ with DNP includes contacting the allograft tissue or organ with a DNP sugar. In some specific examples, the DNP sugar includes N- (2,4-dinitrophenylamino)-acetyl mannosamine (ManDNP-2), tetraacetyl-N-(2,4- dinitrophenylamino) acetyl mannosamine (Ac4ManDNP-2), N-(2,4-dinitrophenylamino)- caprylic mannosamine (ManDNP-6), tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic mannosamine (Ac4ManDNP-6), N-(2,4-dinitrophenylamino)-acetyl galactosamine (GalDNP- 2), tetraacetyl-N-(2,4-dinitrophenylamino) acetyl galactosamine (Ac4Gal-DNP-2), N-(2,4- dinitrophenylamino)-caprylic galactosamine (GalDNP-6), tetraacetyl-N-(2,4- dinitrophenylamino)-caprylic galactosamine (Ac4Gal-DNP-6), N-(2,4-dinitrophenylamino)- acetyl neuraminic acid (Neu5DNP-2), tetraacetyl-N-(2,4-dinitrophenylamino)-acetyl neuraminic acid (Ac4Neu5DNP-2), N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Neu5DNP-6), or tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Ac4Neu5DNP-6). In some aspects, contacting the allograft tissue or organ with the chemical tag (such as a chemical tag that includes DNP) includes immersing the allograft tissue or organ in a solution containing the chemical tag (e.g., a solution containing a DNP sugar) or perfusing the allograft tissue or organ with a solution containing the chemical tag (e.g., a solution containing a DNP sugar). In other aspects, contacting the allograft tissue or organ with the chemical tag (such as a chemical tag containing DNP) includes injecting the allograft tissue or organ with the chemical tag (such as a DNP sugar). The allograft tissue can be injected with the chemical tag at multiple sites, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sites. In some aspects of the disclosed methods, the Treg cells are autologous Treg cells (Tregs obtained from the subject receiving the transplant). In other aspects, the Treg cells are allogeneic Treg cells (obtained from a compatible donor). In yet other aspects, the Treg cells are obtained from universal stem cells. In some aspects, the CAR includes a monoclonal antibody or antigen-binding fragment thereof that specifically binds the chemical tag, a hinge region, a transmembrane7950-111351-02(TM) domain, and one or more intracellular signaling domains. In some examples, the antigen binding fragment is a single chain variable fragment (scFv), such as an scFv that includes in the N-terminal to C-terminal direction a variable light (VL) domain, a peptide linker, and a variable heavy (VH) domain (alternatively, the scFv can have a VH-linker-VL orientation). The peptide linker can be, for example, a glycine-serine rich linker (such as SEQ ID NO: 3). In particular examples, the amino acid sequence of the scFv comprises the amino acid sequence of SEQ ID NO: 5, and optionally the VH domain sequence comprises residues 24-130 of SEQ ID NO: 2 and the VL domain sequence comprises residues 146-266 of SEQ ID NO: 2. In other examples, the antibody or antigen-binding fragment is a single- domain antibody. In some examples, the hinge region includes a CD8 hinge region, such as a mouse or human CD8 hinge region. In specific examples, the amino acid sequence of the CD8 hinge region is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 6. In one example, the amino acid sequence of the CD8 hinge region comprises or consists of SEQ ID NO: 6. In other examples, the hinge region is a CD28 hinge region. In some examples, the transmembrane domain includes a CD28 transmembrane domain, such as a mouse or human CD28 transmembrane domain. In other examples, the transmembrane domain is a CD8 transmembrane domain. In some examples, the one or more intracellular signaling domains includes a CD28 signaling domain, such as a human or mouse CD28 signaling domain. In specific examples, the amino acid sequence of the CD28 transmembrane domain and signaling domain is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 7. In one example, the amino acid sequence of the CD28 transmembrane and signaling domains comprises or consists of SEQ ID NO: 7. In some examples, the one or more intracellular signaling domains includes, or further includes, a CD3ζ signaling domain, such as a human or mouse CD3ζ signaling domain. In specific examples, the amino acid sequence of the CD3ζ signaling domain is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 8. In one example, the amino acid sequence of the CD3ζ signaling domain comprises or consists of SEQ ID NO: 8. In other examples, the one or more intracellular signaling domains includes or further includes a FcεRIγ, 4-1BB (CD137), ICOS, OX40 (CD134), CD27 and / or DAP10 signaling domain.7950-111351-02In some aspects, the Treg cells expressing the chemical tag-specific CAR are produced by transducing the Treg cells with a lentivirus vector encoding the CAR. In some examples, the nucleic acid sequence encoding the CAR is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 1, or comprises or consists of SEQ ID NO: 1. In some examples, the CAR-expressing Treg cells are administered to the subject 1 to 5 days following transplant. In some examples, the subject is administered at least 1, at least 2, at least 3, at least 4 or at least 5 doses of the CAR-expressing Treg cells. The allograft tissue or organ can be any organ or tissue. In some aspects, the allograft tissue or organ includes skin, blood vessel, bone, bone marrow, stem cells, cartilage, tendon, ligament, nerves, cornea, heart valve, heart, intestine, stomach, kidney, liver, lung, pancreas, and / or uterus. Also provided herein are kits for reducing the risk of allograft rejection in a subject. The kit includes a chemical tag and / or a viral vector encoding a CAR that specifically binds the chemical tag. In some aspects, the chemical tag includes dinitrophenyl (DNP) or a DNP analog, fluorescein or a fluorescein derivative, biotin or a biotin derivative, sulfanilamide or a sulfanilamide derivative, a nitrated amino acid, a modified nucleotide or nucleoside, a biocompatible polymer, or an oxidized lipid. In some examples in which the chemical tag is a DNP analog, the chemical tag includes dinitrochlorobenzene (DNCB), 3-nitro-4- hydroxyphenylacetate (NHPA), 2,4-dinitrophenol, picric acid, 2,5-dinitrophenol, m- nitrophenol, 2,6-dinotrophenol, or p-nitroaniline. In some examples in which the chemical tag is a nitrated amino acid, the chemical tag includes nitrotyrosine. In some examples in which the chemical tag is a modified nucleotide or nucleoside, the chemical tag includes 5- bromo-2'-deoxyuridine (BrdU) or 5-methylcytidine. In some examples in which the chemical tag is a biocompatible polymer, the chemical tag includes polyethylene glycol (PEG) or oligo(ethylene glycol) (OEG). In yet other examples in which the chemical tag is an oxidized lipid, the chemical tag includes 4-hydroxynonenal. In some examples, the kit further includes one or more additional components to carry out metabolic labelling of the allograft tissue or organ with the chemical tag, transduction of Treg cells with the viral vector and / or administering the transduced Treg cells to a subject (e.g., instructions, cell culture media, cell culture flasks, tubes, syringes, needles, or any combination thereof). In some aspects of the kits, the chemical tag is DNP in the form of a DNP sugar. In some examples, the DNP sugar includes N-(2,4-dinitrophenylamino)-acetyl mannosamine (ManDNP-2), tetraacetyl-N-(2,4-dinitrophenylamino) acetyl mannosamine (Ac4ManDNP-2),7950-111351-02N-(2,4-dinitrophenylamino)-caprylic mannosamine (ManDNP-6), tetraacetyl-N-(2,4- dinitrophenylamino)-caprylic mannosamine (Ac4ManDNP-6), N-(2,4-dinitrophenylamino)- acetyl galactosamine (GalDNP-2), tetraacetyl-N-(2,4-dinitrophenylamino) acetyl galactosamine (Ac4Gal-DNP-2), N-(2,4-dinitrophenylamino)-caprylic galactosamine (GalDNP-6), tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic galactosamine (Ac4Gal-DNP-6), N-(2,4-dinitrophenylamino)-acetyl neuraminic acid (Neu5DNP-2), tetraacetyl-N-(2,4-dinitrophenylamino)-acetyl neuraminic acid (Ac4Neu5DNP-2), N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Neu5DNP- 6), or tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Ac4Neu5DNP-6). In some aspects of the kits, the viral vector is a lentiviral vector. In some aspects of the kits, the CAR encoded by the viral vector includes a monoclonal antibody or antigen-binding fragment thereof that specifically binds the chemical tag (such as a scFv or a single-domain antibody), a hinge region, a transmembrane domain, and one or more intracellular signaling domains. In some examples, the antigen binding fragment is a scFv that includes, in the N-terminal to C-terminal direction, a VL domain, a peptide linker, and a VH domain. The peptide linker can be, for example, a glycine-serine rich linker (such as SEQ ID NO: 3). In some examples, the hinge region comprises a CD8 hinge region, the transmembrane domain includes a CD28 transmembrane domain and / or the one or more intracellular signaling domains includes a CD28 signaling domain and a CD3ζ signaling domain. IV. Overview of Aspects Aspect 1. A method of reducing allograft rejection or risk of allograft rejection in a subject, comprising: metabolically labeling an allograft tissue or organ with a chemical tag to produce a labelled allograft tissue or organ; transplanting the labelled allograft tissue or organ in the subject; and administering to the subject a therapeutically effective amount of T regulatory (Treg) cells expressing a chimeric antigen receptor (CAR) that specifically binds the chemical tag displayed on the allograft tissue or organ, thereby reducing the allograft rejection or the risk of allograft rejection in the subject.7950-111351-02Aspect 2. The method of aspect 1, wherein the chemical tag comprises dinitrophenyl (DNP) or a DNP analog, fluorescein or a fluorescein derivative, biotin or a biotin derivative, sulfanilamide or a sulfanilamide derivative, a nitrated amino acid, a modified nucleotide or nucleoside, a biocompatible polymer, or an oxidized lipid. Aspect 3. The method of aspect 2, wherein: the DNP analog comprises dinitrochlorobenzene (DNCB), 3-nitro-4- hydroxyphenylacetate (NHPA), 2,4-dinitrophenol, picric acid, 2,5-dinitrophenol, m- nitrophenol, 2,6-dinotrophenol, or p-nitroaniline; the nitrated amino acid comprises nitrotyrosine; the modified nucleotide or nucleoside comprises 5-bromo-2'-deoxyuridine (BrdU) or 5-methylcytidine; the biocompatible polymer comprises polyethylene glycol (PEG) or oligo(ethylene glycol) (OEG); or the oxidized lipid comprises 4-hydroxynonenal. Aspect 4. The method of aspect 1 or aspect 2, wherein the chemical tag comprises DNP. Aspect 5. The method of aspect 4, wherein metabolically labeling the allograft tissue or organ with DNP comprises contacting the allograft tissue or organ with a DNP sugar. Aspect 6. The method of aspect 5, wherein the DNP sugar comprises N-(2,4- dinitrophenylamino)-acetyl mannosamine (ManDNP-2), tetraacetyl-N-(2,4- dinitrophenylamino) acetyl mannosamine (Ac4ManDNP-2), N-(2,4-dinitrophenylamino)- caprylic mannosamine (ManDNP-6), tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic mannosamine (Ac4ManDNP-6), N-(2,4-dinitrophenylamino)-acetyl galactosamine (GalDNP- 2), tetraacetyl-N-(2,4-dinitrophenylamino) acetyl galactosamine (Ac4Gal-DNP-2), N-(2,4- dinitrophenylamino)-caprylic galactosamine (GalDNP-6), tetraacetyl-N-(2,4- dinitrophenylamino)-caprylic galactosamine (Ac4Gal-DNP-6), N-(2,4-dinitrophenylamino)- acetyl neuraminic acid (Neu5DNP-2), tetraacetyl-N-(2,4-dinitrophenylamino)-acetyl neuraminic acid (Ac4Neu5DNP-2), N-(2,4-dinitrophenylamino)-caprylic neuraminic acid7950-111351-02(Neu5DNP-6), or tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Ac4Neu5DNP-6). Aspect 7. The method of aspect 5 or aspect 6, wherein contacting the allograft tissue or organ with the DNP sugar comprises immersing the allograft tissue or organ in a solution containing the DNP sugar or perfusing the allograft tissue or organ with a solution containing the DNP sugar. Aspect 8. The method of aspect 5 or aspect 6, wherein contacting the allograft tissue or organ with the DNP sugar comprises injecting the allograft tissue or organ with the DNP sugar. Aspect 9. The method of any one of aspects 1-8, wherein the Treg cells are autologous Treg cells. Aspect 10. The method of any one of aspects 1-8, wherein the Treg cells are allogeneic Treg cells or Treg cells obtained from universal stem cells. Aspect 11. The method of any one of aspects 1-10, wherein the CAR comprises: a monoclonal antibody or antigen-binding fragment thereof that specifically binds the chemical tag; a hinge region; a transmembrane domain; and one or more intracellular signaling domains. Aspect 12. The method of aspect 11, wherein the antigen binding fragment is a single chain variable fragment (scFv). Aspect 13. The method of aspect 12, wherein the scFv comprises in the N- terminal to C-terminal direction a variable light (VL) domain, a peptide linker, and a variable heavy (VH) domain. Aspect 14. The method of any one of aspects 11-13, wherein: the hinge region comprises a CD8 hinge region;7950-111351-02the transmembrane domain comprises a CD8 transmembrane domain; and / or the one or more intracellular signaling domains comprise a CD28 signaling domain and a CD3ζ signaling domain. Aspect 15. The method of any one of aspects 1-14, wherein the Treg cells expressing the chemical tag-specific CAR are produced by transducing the Treg cells with a lentivirus vector encoding the CAR. Aspect 16. The method of any one of aspects 1-14, wherein the CAR-expressing Treg cells are administered to the subject 1 to 5 days following transplant. Aspect 17. The method of aspect 16, wherein the subject is administered at least 1, at least 2, at least 3, at least 4 or at least 5 doses of the CAR-expressing Treg cells. Aspect 18. The method of any one of aspects 1-17, wherein the allograft tissue or organ comprises skin, blood vessel, bone, bone marrow, stem cells, cartilage, tendon, ligament, nerves, cornea, heart valve, heart, intestine, stomach, kidney, liver, lung, pancreas, or uterus. Aspect 19. A kit, comprising: a chemical tag, wherein the chemical tag comprises dinitrophenyl (DNP) or a DNP analog, fluorescein or a fluorescein derivative, biotin or a biotin derivative, sulfanilamide or a sulfanilamide derivative, a nitrated amino acid, a modified nucleotide or nucleoside, a biocompatible polymer, or an oxidized lipid; a viral vector encoding a chimeric antigen receptor (CAR) that specifically binds the chemical tag; engineered CAR T regulatory (Treg) cells specific for the chemical tag; and / or instructions, a Treg isolation kit, cell culture media, cell culture flasks, tubes, syringes, needles, or any combination thereof. Aspect 20. The kit of aspect 19, wherein the chemical tag comprises DNP in the form of a DNP sugar.7950-111351-02Aspect 21. The kit of aspect 20, wherein the DNP sugar comprises N-(2,4- dinitrophenylamino)-acetyl mannosamine (ManDNP-2), tetraacetyl-N-(2,4- dinitrophenylamino) acetyl mannosamine (Ac4ManDNP-2), N-(2,4-dinitrophenylamino)- caprylic mannosamine (ManDNP-6), tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic mannosamine (Ac4ManDNP-6), N-(2,4-dinitrophenylamino)-acetyl galactosamine (GalDNP- 2), tetraacetyl-N-(2,4-dinitrophenylamino) acetyl galactosamine (Ac4Gal-DNP-2), N-(2,4- dinitrophenylamino)-caprylic galactosamine (GalDNP-6), tetraacetyl-N-(2,4- dinitrophenylamino)-caprylic galactosamine (Ac4Gal-DNP-6), N-(2,4-dinitrophenylamino)- acetyl neuraminic acid (Neu5DNP-2), tetraacetyl-N-(2,4-dinitrophenylamino)-acetyl neuraminic acid (Ac4Neu5DNP-2), N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Neu5DNP-6), or tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Ac4Neu5DNP-6). Aspect 22. The kit of any one of aspects 19-21, wherein the viral vector is a lentiviral vector. Aspect 23. The kit of any one of aspects 19-22, wherein the CAR comprises: a monoclonal antibody or antigen-binding fragment thereof that specifically binds the chemical tag; a hinge region; a transmembrane domain; and one or more intracellular signaling domains. Aspect 24. The kit of aspect 23, wherein: the antigen binding fragment is a single chain variable fragment (scFv); the hinge region comprises a CD8 hinge region; the transmembrane domain comprises a CD8 transmembrane domain; and / or the one or more intracellular signaling domains comprise a CD28 signaling domain and a CD3ζ signaling domain. Aspect 25. The kit of any one of aspects 19-24, wherein the engineered CAR Treg cells are obtained from stem cells or an allogeneic source.7950-111351-02EXAMPLES The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified. Example 1: Materials and Methods This example describes the materials and experimental procedures used for the studies described in Examples 2-8. Materials and Instrumentation D-Mannosamine hydrochloride, N-(2,4-Dinitrophenyl) glycine, Succinimidyl-N-(2,4- dinitrophenyl)-6-aminocaproate, acetic anhydride, MTT, and other chemical reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise noted. Ovalbumin DNP conjugate (DNP-OVA) was purchased from Nanocs (New York, NY, USA). Fetal Bovine Serum (FBS) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). IL-2 was purchased from PeproTech (Cranbury, NJ, USA). CellTrace™ CFSE Cell Proliferation Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies used in this study include fluorophore-conjugated anti-CD3 (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD25 (PC61.5), anti-CD19 (eBio1D3), anti- CD11b (M1 / 70), anti-CD11c (N418), anti-CD69 (H1.2F3), anti-CD80 (16-10A1), anti-CD86 (GL1), anti-Helios (22F6), and anti-HA tag (RM305). All antibodies were diluted according to the manufacturer’s guidelines. Proteome Profiler Mouse Cytokine Array Kit, Panel A was purchased from R&D Systems (Minneapolis, MN, USA). High-performance liquid chromatography (HPLC) analysis was performed on a Shimadzu CBM-20A system (Shimadzu, Kyoto, Japan) equipped with an SPD-20A PDA detector (190–800 nm), an RF- 20A fluorescence detector, and an analytical C18 column (Shimadzu, 3 μm, 50 × 4.6 mm, Kyoto, Japan). Preparative HPLC was performed on a CombiFlash®Rf system (Teledyne ISCO, Lincoln, NE, USA) equipped with a RediSep®Rf HP C18 column (Teledyne ISCO, 30 g, Lincoln, NE, USA). Lyophilization was conducted in a Labconco FreeZone lyophilizer (Kansas City, MO, USA). Nuclear Magnetic Resonance (NMR) spectra were recorded on a Varian U500 (500 MHz) or VXR500 (500 MHz), or a Bruker Carver B500 (500 MHz) spectrometer. Electrospray ionization (ESI) mass spectra were obtained from a Waters ZMD Quadrupole Instrument (Waters, Milford, MA, USA). Flow cytometry was performed on a BD FACSymphony™ A1 Cell Analyzer (BD, Franklin Lakes, NJ, USA) or Attune NxT flow7950-111351-02cytometers and analyzed on FCS Express v6. Fluorescence was also measured on a Biotek plate reader (BioTek Instruments, Winooski, VT, USA). Frozen tissues were embedded with optimum cutting temperature (OCT) compound (Sakura Finetek USA, Torrance, CA, USA), sectioned by a Leica CM3050S Cryostat, and mounted onto glass slides. Mice images are taken with a Canon EOS 5D Mark IV DSLR Camera. Cell lines and animals Human primary epidermal keratinocytes, CHO, HEK293, LS174T, A549, B16F10, MSC, and EL4 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). ID8-Cos and ID8 cell lines were kind gifts from Prof. David Kranz’s lab at the University of Illinois at Urbana-Champaign. Human primary epidermal keratinocytes were cultured in dermal cell basal medium (ATCC PCS-200-030) supplemented with a keratinocyte growth kit (ATCC PCS-200-040). CHO and A549 cells were cultured with Kaighn's Modification of Ham's F-12 media containing 10% FBS, 100 units / mL Penicillin G and 100 μg / mL streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 humidified incubator. HEK293, EL4, B16F10, ID8-Cos, ID8, MSC, and LS174T cells were cultured with DMEM media containing 10% FBS, 100 units / mL Penicillin G and 100 μg / mL streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2humidified incubator. Female C57BL / 6 and Balb / c mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Feed and water were available ad libitum. Artificial light was provided in a 12 h / 12 h cycle. All procedures involving animals were done in compliance with National Institutes of Health and Institutional guidelines with approval from the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Urbana-Champaign. Synthesis of DNP-NHS To a solution of N-(2,4-dinitrophenyl) glycine (1.20 g, 5.0 mmol) in DMF (8 mL) was added N,N'-dicyclohexylcarbodiimide (1.03 g, 5.0 mmol) and N-hydroxysuccinimide (0.58 g, 5.0 mmol). The mixture was stirred at room temperature for 18 h. The precipitate was filtered out and washed with DMF (2 mL). DMF was then removed to yield a yellow solid. The crude product was recrystallized 3 times with DCM at 0°C to obtain an off-white solid (1.31g, yield: 77.8%).1H NMR (500 MHz, CDCl3) δ 9.17 (d, J = 2.7 Hz, 1H), 8.97 (d, J = 5.4 Hz, 1H), 8.33 (dd, J = 9.4, 2.7 Hz, 1H), 6.82 (d, J = 9.4 Hz, 1H), 4.24 (d, J = 5.2 Hz, 2H), 3.90 (s, 4H).7950-111351-02Synthesis of ManDNP-2 To a solution of D-Mannosamine hydrochloride (0.54 g, 2.5 mmol) and triethylamine (0.34 mL, 2.5 mmol) in anhydrous methanol was added DNP-NHS (0.94 g, 2.8 mmol). The mixture was stirred at room temperature for 18 h. Then the solvent was evaporated and the crude product was purified by silica gel column chromatography to afford ManDNP-2 as a light yellow solid (0.87g, yield: 86.4%) (3 / 7 α / β isomers).1H NMR (500 MHz, CD3OD_SPE) δ 9.05 (d, J = 2.8 Hz, 1H), 8.30 (dd, J = 9.5, 2.7 Hz, 1H), 7.05 (dd, J = 25.8, 9.5 Hz, 1H), 5.04 & 4.90 (d, J = 1.5 Hz, 1H), 4.33 & 4.28 (dd, J = 4.6, 1.7 Hz, 1H), 4.25 (d, J = 4.3 Hz, 2H), 4.03 & 3.68 (dd, J = 9.7, 4.6 Hz, 1H), 3.89 – 3.74 (m, 4H), 3.56 & 3.46 (t, J = 9.8 Hz, 1H).13C NMR (126 MHz, CD3OD_SPE) δ 166.97, 153.88, 131.49, 124.90, 115.40, 96.91, 72.06, 69.13, 67.15, 60.19, 59.98, 55.35, 54.83, 45.62. HRMS (ESI) Calcd for C14H18N4O10Na [M+Na]+: 425.0921; Found: 425.0909. Synthesis of Ac4ManDNP-2 To a solution of ManDNP-2 (0.80 g, 2 mmol) in pyridine was added acetic anhydride (1.22 g, 12 mmol) dropwise, and the reaction mixture was stirred at room temperature for 18 h. The reaction with then quenched by methanol and stirred for another 2 h. Then the solvent was evaporated and the crude product was purified by silica gel column chromatography to afford Ac4ManDNP-2 as a light yellow solid (0.83 g, yield: 72.5%) (4 / 6 α / β isomers).1H NMR (500 MHz, CDCl3) δ 9.11 (s, 1H), 8.95 (d, J = 11.3 Hz, 1H), 8.28 (dd, J = 9.3, 2.6 Hz, 1H), 6.78 (dd, J = 9.4, 6.4 Hz, 1H), 6.27 (dd, J = 32.9, 8.9 Hz, 1H), 5.89 (d, J = 71.9 Hz, 1H), 5.28 (dd, J = 10.2, 4.3 Hz, 1H), 4.99 (dt, J = 20.2, 10.0 Hz, 2H), 4.81 – 4.55 (m, 1H), 4.09 – 3.84 (m, 2H), 2.13 (s, 2H), 2.08 – 1.85 (m, 12H).13C NMR (126 MHz, CDCl3) δ 170.47, 169.96, 169.83, 169.59, 168.09, 168.01, 167.51, 167.20, 147.31, 138.35, 132.41, 130.72, 124.53, 114.96, 90.88, 89.82, 72.94, 71.86, 70.27, 68.96, 65.10, 64.87, 62.38, 61.70, 51.82, 49.92, 45.90, 20.07, 18.34. HRMS (ESI) Calcd for C22H27N4O14 [M + H]+: 571.1524; Found: 571.1527. Synthesis of ManDNP-6 To a solution of D-Mannosamine hydrochloride (0.54 g, 2.5 mmol) and triethylamine (0.34 mL, 2.5 mmol) in anhydrous methanol was added Succinimidyl-N-(2,4-dinitrophenyl)- 6-aminocaproate (1.11 g, 2.8 mmol). The mixture was stirred at room temperature for 18 h. Then the solvent was evaporated and the crude product was purified by silica gel column7950-111351-02chromatography to afford ManDNP-6 as a light yellow solid (0.86 g, yield: 75.6%).1H NMR (500 MHz, CD3OD) δ 8.96 (d, J = 2.7 Hz, 1H), 8.23 (dd, J = 9.6, 2.7 Hz, 1H), 7.11 (dd, J = 9.6, 5.0 Hz, 1H), 4.94 (d, J = 1.3 Hz, 1H), 4.83 (d, J = 1.4 Hz, 1H), 4.24 (dd, J = 4.7, 1.4 Hz, 1H), 3.96 (dd, J = 9.7, 4.7 Hz, 1H), 3.83 – 3.76 (m, 1H), 3.72 (ddt, J = 8.0, 5.9, 2.4 Hz, 2H), 3.54 (t, J = 9.7 Hz, 1H), 3.44 (t, J = 7.2 Hz, 1H), 3.30 (s, 1H), 2.27 (dtt, J = 21.5, 14.2, 7.4 Hz, 1H), 1.70 (dp, J = 30.4, 7.4 Hz, 5H), 1.46 (ddt, J = 16.2, 10.2, 6.0 Hz, 3H).13C NMR (126 MHz, CD3OD_SPE) δ 175.28, 148.33, 135.43, 129.68, 123.37, 114.37, 93.61, 76.82, 72.01, 69.21, 67.06, 60.80, 53.63, 42.72, 35.26, 28.05, 26.04, 25.08. HRMS (ESI) Calcd for C18H26N4O10Na [M + Na]+ 481.1547; Found: 481.1545. Synthesis of Ac4ManDNP-6 To a solution of ManDNP-6 (0.91 g, 2 mmol) in pyridine was added acetic anhydride (1.22 g, 12 mmol) dropwise, and the reaction mixture was stirred at room temperature for 18 h. The reaction with then quenched by methanol and stirred for another 2 h. Then the solvent was evaporated and the crude product was purified by silica gel column chromatography to afford Ac4ManDNP-6 as a light yellow solid (0.77 g, yield: 62.0%).1H NMR (500 MHz, CDCl3) δ 9.06 (d, J = 2.7 Hz, 1H), 8.51 (t, J = 5.3 Hz, 1H), 8.20 (dd, J = 9.3, 2.7 Hz, 1H), 7.55 (ddd, J = 86.9, 5.7, 3.3 Hz, 1H), 6.87 (d, J = 9.3 Hz, 1H), 5.96 (s, 1H), 5.79 (t, J = 9.0 Hz, 1H), 5.26 (dd, J = 10.1, 4.4 Hz, 1H), 5.11 (t, J = 10.3 Hz, 1H), 4.60 (dd, J = 9.4, 4.4 Hz, 1H), 4.22 (dt, J = 12.4, 6.2 Hz, 1H), 4.17 – 3.99 (m, 3H), 3.38 (q, J = 6.6 Hz, 2H), 2.07 – 1.86 (m, 12H), 1.49 (dh, J = 14.6, 6.5 Hz, 2H), 1.19 (t, J = 7.1 Hz, 2H), 0.84 (dt, J = 12.6, 7.0 Hz, 2H).13C NMR (126 MHz, CDCl3) δ 171.67, 170.15, 169.43, 168.93, 168.77, 167.16, 147.33, 134.96, 129.30, 124.15, 112.93, 90.65, 69.16, 68.01, 67.13, 64.93, 61.09, 59.38, 48.21, 41.07, 35.34, 35.14, 27.90, 25.39, 24.05, 19.76, 13.03, 9.30. HRMS (ESI) Calcd for C26H35N4O14 [M + H]+: 627.2131; Found: 627.2131. Flow cytometry analysis of DNP-labeled cells Cells were incubated with DNP-sugars at 37°C for 72 h. After washing with PBS three times, cells were lifted with trypsin, suspended in fresh FACS buffer, stained with CF®640R-conjugated DNP polyclonal antibody for 30 min, and analyzed by a flow cytometer.7950-111351-02General procedures for flow cytometry analysis of immune cells After the designed treatments, cells were stained with fluorophore-conjugated antibodies and live / dead fixable stain in FACS buffer (1X PBS buffer supplemented with 5% FBS), washed with FACS buffer for three times, and stored in FACS buffer containing 0.4% PFA at 4oC prior to flow cytometry analysis. For the staining of transcription factors such as Foxp-3, cells were first stained with live / dead stain and antibodies for surface markers, treated with eBioscience™ Foxp3 Transcription Factor Staining Buffer Set, and then stained with fluorophore-conjugated anti-Foxp3, prior to flow cytometry analysis. For isolated blood cells from mice, cells were treated with the ACK lysis buffer for 2 min at room temperature to remove red blood cells before staining. Lentivirus construction and production HEK293T cells with low passage numbers were used for lentiviral packaging. One day before transfection, HEK293T cells were plated into 12-well plates.1 μg of mixed plasmids (0.5 μg CAR-modified pLJM1 (Addgene #19319), 0.45 μg pCMV-dR8.2 dvpr (Addgene #8455), and 0.05 μg pCMV-VSV-G (Addgene #8454) in 50 μL was incubated with 3 μL FuGene HD for 5 min at room temperature and added to cells. After 24 h, the media will be changed to DMEM containing 30% FBS. At 60 h, the supernatant was collected and the cell debris was removed via centrifugation at 4000 rpm for 5 min. The supernatant was aliquoted to 200 μL per vial, and the viral titer was measured via the standard RT-qPCR method using a qPCR lentivirus titer kit (Abmgood, Richmond, BC, Canada). The virus aliquots were stored at -80oC before further use. CAR Treg transduction Primary T cells were isolated from the spleen of C57BL / 6 mice and magnetically selected using the CD4+ / CD25+T cell isolation kit (Miltenyi Biotec) to collect CD4+CD25+T cells. The isolated CD4+CD25+T cells were activated with Dynabead (Thermo Fisher Scientific 111161D) at 1:1 number ratio. After 48 h, 105activated T cells were plated into 12- well plates spiked with 8 μg / mL polybrene (Sigma-Aldrich), and transduced by adding a pre- titrated volume (~50 μL) of pre-warmed lentiviral supernatant containing the CAR construct. After 24 h, the lentivirus was removed by centrifugation at 350g for 5 min. The transduced T cells were then cultured for 72 h in the presence of 2 μg / mL puromycin for CAR Treg selection. After the removal of Dynabeads, CAR Tregs were used or cryopreserved in FBS containing 10% DMSO. The transduction efficiency was determined by measuring the7950-111351-02expression of the HA tag via flow cytometry. For mouse studies, CAR Tregs were restimulated and expanded for three days before injections. Stimulation of CAR Tregs by DNP-OVA 105anti-DNP CAR Tregs, anti-CD19 CAR Tregs, or naïve Tregs were plated into 12- well plates, and treated with different concentrations of DNP-OVA for 24 h. Cells without DNP-OVA treatment were used as controls. Tregs were collected and washed three times to remove unbounded DNP-OVA. Cells were then stained with fluorophore-conjugated anti- DNP, anti-Helios, or anti-CD69, prior to flow cytometry analysis. Suppression of T cells by CAR Tregs Proliferation assay: 3×104CHO cells were incubated with ManDNP-6 (50 μM) for three days and rinsed. CD8+T cells were isolated from the spleen of C57BL / 6 mice and pre- stimulated with anti-CD3 / CD28 Dynabeads and IL-2 for 24 h. After the removal of Dynabeads, CD8+T cells were stained with CFSE and co-incubated with CAR Tregs at different Treg: CD8+T ratios in the presence or absence of DNP-labeled CHO cells for three days, before flow cytometry analysis Tumor cell lysis assay: 3×104E.G7-OVA cells were plated into 12-well plates and incubated with ManDNP-6 (50 μM) or PBS for three days. After washing, E.G7-OVA cells were stained with Calcein AM (Invitrogen #C1430) for 30 min at 37°C. OT-1 cells and E.G7- OVA cells were mixed and seeded into 96-well U-bottom plates (E:T ratio = 1:2). CAR Tregs were then added into each well at different Treg:OT1 ratios. Cells were incubated at 37°C for 6 h. E.G7-OVA cells treated with 1% Triton X-100 were used as positive controls. Fluorescence measurement of supernatants was performed on a Biotek plate reader at λex = 485 nm and λem= 528 nm. The cytolysis efficiency was calculated as (E528,sample− E528,NC) / (E528, PC − E528, NC) ×100%, where E528, PC and E528, NC are the emission intensity of positive and negative controls, respectively at λem = 528 nm. Suppression of DCs by CAR Tregs Bone marrow cells were extracted from the femur and tibia of C57BL / 6 mice and cultured in RPMI-1640 full media supplemented with 20 ng / mL GM-CSF for six days to enrich bone marrow-derived dendritic cells (BMDCs).3×104CHO cells were plated into 12- well plates and incubated with ManDNP-6 (50 μM) or PBS for three days. After washing,7950-111351-02BMDCs and Tregs were added to CHO cells and co-incubated for four days. CD86 / CD80 expression of BMDCs was determined via flow cytometry analyses. Skin transplantation A major histocompatibility (MHC)-mismatched murine skin allotransplantation model was used to evaluate the mitigation of immune rejection by adoptively transferred CAR Tregs. Full-thickness skin tissues (1.0 cm × 1.5 cm) from fur-shaven BALB / c donors were harvested at the level of the areolar connective tissue with panniculus carnosus tissues carefully removed using blunt-tipped forceps. The fur of the recipient C57BL / 6 mice was shaven, and a 1.0 cm by 1.5 cm skin at the dorsal trunk was resected while preserving the panniculus carnosus and surrounding blood vessels. Before transplantation, an aqueous solution of ManDNP-6 (10 mg / mL) was injected into the skin graft at multiple locations. The skin graft was then transplanted and sutured to the surrounding endogenous skin. The transplantation area was protected with a dry gauze and bandage for 7 days. Mice were administered with carprofen (5 mg / kg) once before the surgery and three times after the surgery (every 24 h).5.0 × 105CAR Tregs with C57BL / 6 background were intravenously injected at 2, 3, and 4 days (3 doses in total). For in vivo Treg stimulation studies, skin allografts were harvested at 7 days for immune cell analysis. For survival studies, grafts were monitored daily for erythema, erosion, contraction, or necrosis of the allografts. Animal survival was also closely monitored. Statistical analysis Statistical analysis was performed using GraphPad Prism v6 and v8. Sample variance was tested using the F test. For samples with equal variance, the significance between the groups was analyzed by a two-tailed student’s t-test. For samples with unequal variance, a two-tailed Welch’s t-test was performed. For multiple comparisons, a one-way analysis of variance (ANOVA) with a post hoc Fisher’s LSD test was used. The results were deemed significant at 0.01 < *P ≤ 0.05, highly significant at 0.001 < **P ≤ 0.01, and extremely significant at ***P ≤ 0.001. Example 2: Metabolic labeling of cells with DNP To test the feasibility of labeling cells with DNP groups, four types of DNP-sugars were synthesized: N-(2,4-dinitrophenylamino)-acetyl mannosamine (ManDNP-2), tetraacetyl-N-(2,4-dinitrophenylamino) acetyl mannosamine (Ac4ManDNP-2), N-(2,4-7950-111351-02dinitrophenylamino)-caprylic mannosamine (ManDNP-6), and tetraacetyl-N-(2,4- dinitrophenylamino)-caprylic mannosamine (Ac4ManDNP-6) (FIGS.2A, 6A-6E, 7A-7E). ID8-Cos cells were incubated with DNP-sugars or PBS for three days, followed by the detection of cell-surface DNP with AF647-conjugated anti-DNP. Compared to control cells, cells treated with ManDNP-6 or Ac4ManDNP-6 showed significantly higher AF647 fluorescence intensity, indicating the successful metabolic labeling of cells with DNP groups (FIGS.2B-2C). Similarly, ManDNP-6 and Ac4ManDNP-6 also successfully labeled B16F10 cells (FIGS.8A-8B) and ID8 cells (FIGS.8D-8E) in a concentration-dependent manner. In comparison with ManDNP-6 or Ac4ManDNP-6, ManDNP-2 and Ac4ManDNP-2 showed a much lower metabolic labeling efficiency for ID8-Cos, B16F10, and ID8 cancer cells (FIGS. 2D-2E, 8C, 8F). For non-cancerous primary human keratinocytes and Chinese hamster ovary (CHO) cells, ManDNP-6 also managed to label cells with DNP groups (FIGS.2F-2G, 9A- 9B). Considering its favorable metabolic labeling effect and water-solubility, ManDNP-6 was used for subsequent experiments. ManDNP-6 consistently showed successful labeling of other cancerous cells including LS174T colon cancer cells, A549 lung cancer cells, and EL-4 lymphoma cells, and non-cancerous cells including HEK293 kidney cells and mouse mesenchymal stem cells (MSCs) (FIGS.2H-2L, 9C-9G). The replenishment of DNP-sugar- containing media during the 72-h cell culture time failed to improve the metabolic labeling efficiency (FIG.9H). However, by starving the cells with low-serum (0.5% FBS) media for 12 or 24 h during DNP-sugar treatment, the labeling efficiency was significantly enhanced (FIG.9I), providing a viable strategy to improve the overall metabolic labeling efficiency. Example 3: Development of anti-DNP CAR Tregs After demonstrating the successful metabolic labeling of cells with DNP groups, anti- DNP CAR Tregs were engineered. A universal second-generation anti-DNP CAR construct that includes an extracellular anti-DNP scFv in the form of VL-(GGGGS)3-VH (SEQ ID NO: 3) (Gonzalez and Waxman, Clin Immunol 100:362-371, 2001; White et al., Mol Immunol 33:759-768, 1996), a transmembrane CD8 domain, and intracellular CD28 / CD3ζ signaling domains was designed (FIGS.3A, 10A, 10C and 17). The nucleotide and amino acid sequences of the CAR construct are provided below and set forth herein as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. NotI-leader-DNP VL-(G4S)3Linker-DNP VH-CD8 Hinge-CD28-CD3ζ-STOP- EcoRI7950-111351-02Nucleotide sequence (SEQ ID NO: 1) GCGGCCGCCACCATGGGATGGAGTTGTATCATTCTCTTCCTCGTCGCTACCGCCACCGGAGTGCATTCAgatat ccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagtt gcagggcaagtcaggatattagcaattatttaaactggtatcagcagaaaccagatggaact gttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtgg cagtgggtctggaacagattattctctcaccattagcaacctggaacaagaagatattgcca cttacttttgccaacagggtaatacgcttccgtggacgttcggtggaggcaccaagctggaa atcaaaggcggcgggggttctggtggcggcggcagcggcggtggaggatcagatgtacgcct tcaggagtcaggacctggcctcgtgaaaccttctcagtctctgtctctcacctgctctgtca ctggctactccatcaccaatagttattactggaactggattcggcagtttccaggaaacaaa ctggaatggatggtctacataggctacgacggtagcaataactacaacccatctctcaaaaa tcgaatctccatcactcgtgacacatctaagaaccagtttttcctgaagttgaactctgtga ctactgaggacacagctacatattactgtgcaagagctacctactatggtaactacaggggg tttgcttactggggccaagggactctggtcactgtctctgcaCTCGAGAAGGTGAACAGCACTACCACAAAACCAGTCCTGAGAACTCCCAGCCCTGTGCATCCAACCGGGACATCCCAGCCACAGCGACCAGAGGATTGCCGACCACGAGGATCCGTGAAGGGAACCGGGCTGGACTTCGCCTGTGATTCCTCTCCTAAACTGTTTTGGGCTCTC GTGGTCGTGGCAGGAGTGCTGTTTTGCTACGGGCTGCTCGTCACTGTGGCCCTGTGCGTGATTTGGACCAACAGC AGGAGAAATCGGCTGCTCCAAGTGACTACCATGAACATGACCCCTCGGCGCCCAGGCCTGACAAGAAAGCCCTAC CAGCCTTATGCCCCAGCTCGGGACTTCGCAGCATATCGACCAGCACACGCACGAGCTAAATTTTCTAGGAGTGCTGAAACAGCTGCAAACCTGCAGGATCCCAATCAGCTCTACAACGAGCTGAATCTCGGACGAAGGGAGGAATATGACGTGCTGGAAAAGAAACGAGCTAGGGATCCTGAGATGGGAGGCAAGCAGCAGAGACGGCGCAACCCACAGGAAGGC GTGTACAATGCACTGCAGAAGGACAAAATGGCAGAAGCCTATAGCGAGATTGGAACTAAGGGGGAGCGAAGGAGA GGTAAAGGCCATGATGGTCTGTACCAGGGCCTGAGCACAGCCACAAAGGATACATACGATGCCCTCCACATGCAG ACACTCGCACCCAGGTGATGAGAATTC Feature Nucleotides of SEQ ID NO: 1 NotI site 1-8 AAATMGWSCIILFLVATATGVHSDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNW YQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNT LPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVRLQESGPGLVKPSQSLSLTCSVTGYS ITNSYYWNWIRQFPGNKLEWMVYIGYDGSNNYNPSLKNRISITRDTSKNQFFLKLNS VTTEDTATYYCARATYYGNYRGFAYWGQGTLVTVSALEKVNSTTTKPVLRTPSPVH PTGTSQPQRPEDCRPRGSVKGTGLDFACDSSPKLFWALVVVAGVLFCYGLLVTVAL CVIWTNSRRNRLLQVTTMNMTPRRPGLTRKPYQPYAPARDFAAYRPAHARAKFSRS AETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNA LQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR**EF Feature Amino Acids of SEQ ID NO: 2 7950-111351-02Feature Amino Acids of SEQ ID NO: 2 Linker sequence 131-145 NO: 4) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKGGGGSGGGG SGGGGSDVRLQESGPGLVKPSQSLSLTCSVTGYSITNSYYWNWIRQFPGNKLEWMV YIGYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCARATYYGNYRG FAYWGQGTLVTVSALEKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTG LDFACDSSPKLFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQVTTMNMTPR RPGLTRKPYQPYAPARDFAAYRPAHARAKFSRSAETAANLQDPNQLYNELNLGRRE EYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRG KGHDGLYQGLSTATKDTYDALHMQTLAPR DNP VL-linker-VH scFv (SEQ ID NO: 5) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGV PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKGGGGSGGGG SGGGGSDVRLQESGPGLVKPSQSLSLTCSVTGYSITNSYYWNWIRQFPGNKLEWMV YIGYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCARATYYGNYRG FAYWGQGTLVTVSA CD8 hinge (SEQ ID NO: 6) KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACD CD28 TM and signaling domain (SEQ ID NO: 7) SSPKLFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQVTTMNMTPRRPGLTR KPYQPYAPARDFAAYRPAHA CD3ζ signaling domain (SEQ ID NO: 8) RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNP QEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTL APR To verify the specificity of the anti-DNP CAR towards DNP, a mismatched anti- CD19 CAR with the same transmembrane and signaling domains was cloned (FIGS.10B, 10D). Primary CD4+CD25+Tregs isolated from the spleen of C57BL / 6 mice were transduced via lentiviral vectors to generate anti-DNP CAR Tregs or anti-CD19 CAR Tregs (FIGS.10E- 10F). The expression of CAR on Tregs was detected by AF700-conjugated anti-HA, which showed successful transfection of >60% of Tregs with anti-DNP CAR (FIGS.3B-3C). Similarly, Tregs were also successfully transfected with anti-CD19 CAR (FIG.10G).7950-111351-02To test the binding of anti-DNP CAR Tregs to DNP groups, cells were incubated with DNP-OVA, followed by the detection of surface-bound DNP with AF647-conjugated anti- DNP (FIG.3D). As a result, anti-DNP CAR Tregs, but not anti-CD19 CAR Tregs, showed significantly higher AF647 fluorescence intensity (FIG.3E-3F), indicating the successful capture of DNP-OVA by anti-DNP CAR Tregs. It was also tested whether DNP-OVA binding can induce the activation of anti-DNP CAR Tregs by incubating Tregs with DNP- OVA for three days and detecting the cell-surface Helios, a marker for Treg activation and linage stability (Akimova et al., PloS One 6:e24226, 2011; Takatori et al., Arthritis Rheumatol 67:1491-1502, 2015; Lam et al., Eur J Immunol 52:75-84, 2022). Compared to control cells without DNP-OVA treatment, anti-DNP CAR Tregs treated with DNP-OVA showed an upregulated expression of Helios (FIGS.3G-3H, 11A-11B). In contrast, anti- CD19 CAR Tregs did not show any response towards DNP-OVA (FIGS.3G-3H, 11A-11B). The Helios expression level of anti-DNP CAR Tregs increased with the concentration of DNP-OVA (FIGS.3I-3J, 11C-11D). In addition, DNP-OVA was also able to induce the upregulated expression of CD69, another common activation marker for T cells, on anti-DNP CAR Tregs but not anti-CD19 CAR Tregs (FIG.12A). DNP-OVA stimulation also downregulated the overall secretion of inflammatory cytokines by anti-DNP CAR Tregs (FIG.12B). These experiments demonstrated that anti-DNP CAR Tregs can specifically recognize DNP and can be stimulated upon the recognition of DNP. Example 4: Anti-DNP CAR Tregs can recognize and become activated by DNP-labeled cells It was next studied whether anti-DNP CAR Tregs can recognize and become stimulated by DNP-labeled cells. CHO cells pretreated with ManDNP-6 or PBS were incubated with anti-DNP CAR Tregs or anti-CD19 CAR Tregs at a 1:1 ratio for 24 h (FIG. 4A). Compared to Tregs incubated with unlabeled CHO cells, anti-DNP CAR Tregs incubated with DNP-labeled CHO cells exhibited an elevated expression of Helios (FIGS. 4B-4C), indicating the stimulation of anti-DNP CAR Tregs as a result of DNP recognition. In contrast, anti-CD19 CAR Tregs incubated with DNP-labeled or unlabeled CHO cells showed negligible differences in Helios expression (FIGS.4B-4C). Consistently, anti-DNP CAR Tregs incubated with DNP-labeled CHO cells also exhibited a significantly higher expression of CD69 and CTLA-4 (FIGS.13A-13D), substantiating DNP-mediated stimulation of anti- DNP CAR Tregs. Compared to untreated anti-DNP CAR Tregs, anti-DNP CAR Tregs incubated with DNP-labeled CHO cells also showed increased secretion of anti-inflammatory7950-111351-02cytokines such as IL-4 and IL-10 and decreased secretion of pro-inflammatory cytokines including TNF-α, G-CSF, M-CSF, GM-CSF, IL-6, and IL-12 (FIG.13E). These experiments demonstrated that anti-DNP CAR Tregs can specifically bind to and become stimulated by DNP-labeled cells. Example 5: Anti-DNP CAR Tregs can suppress CD8+T cells Another study was performed to determine whether anti-DNP CAR Tregs could suppress the activity of CD8+T cells in a DNP-specific manner. CFSE-stained mouse CD8+T cells were co-cultured with anti-DNP CAR Tregs in the presence or absence of DNP- labeled CHO cells for three days, followed by the analysis of CD8+T cell proliferation via flow cytometry (FIG.4D). At a Treg / CD8+T ratio of 1:4, anti-DNP CAR Tregs failed to show a noticeable suppressive effect against CD8+T cells (FIGS.4D-4E). In contrast, in the presence of DNP-labeled CHO cells, anti-DNP CAR Tregs showed significant inhibition of CD8+T cell proliferation (FIGS.4D-4E). At a Treg / CD8+T ratio of 1:1, anti-DNP CAR Tregs, with or without stimulation by DNP-labeled CHO cells, were able to suppress the proliferation of CD8+T cells (FIGS.4D-4E). In the presence of DNP-labeled CHO cells, though, anti-DNP CAR Tregs exhibited a significantly enhanced suppressive effect against CD8+T cells (FIGS.4D-4E). To further confirm the CD8+T cell suppression effect of anti- DNP CAR Tregs and assess their protective effect towards DNP-labeled cells, we labeled E.G7-OVA cancer cells with DNP groups via the treatment with ManDNP-6 and incubated them with a mixture of anti-DNP CAR Tregs and SIINFEKL (SEQ ID NO: 9)-specific OT-1 cells. Compared to unlabeled E.G7-OVA cells, DNP-labeled E.G7-OVA cells showed a reduced lysis rate by OT-1 cells, demonstrating the protective effect of anti-DNP CAR Tregs towards DNP-labeled cells (FIG.4F). In contrast, anti-CD19 CAR Tregs did not exhibit any protective effect towards DNP-labeled E.G7-OVA cells (FIG.4F). Example 6: Anti-DNP CAR Tregs can suppress dendritic cells In addition to suppressing the activity of T cells, recent studies raised a growing appreciation for the role of activated Tregs in the suppression of antigen presenting cells, especially dendritic cells (DCs), through CTLA-4 mediated signaling (Walker and Sansom, Nat Rev Immunol 11:852-863, 2011; Tai et al., Blood 119:5155-5163, 2012; Wang et al., Cancer Immunol Immunother 60:381-388, 2011). The ability of anti-DNP CAR-Tregs to suppress dendritic cells in a DNP-specific manner was therefore assessed. Anti-DNP CAR Tregs or anti-CD19 CAR Tregs were incubated with bone marrow derived DCs, in the7950-111351-02presence of DNP-labeled CHO cells, for four days. At a Treg / DC ratio of 1:1, anti-CD19 CAR Tregs slightly downregulated the expression of CD86 and CD80, two well-known activation markers, by DCs (FIGS.4G-4H, 14A-14B). In comparison with anti-CD19 CAR Tregs, anti-DNP CAR Tregs significantly reduced the expression levels of CD86 and CD80 on DCs (FIGS.4G-4H, 14A-14B). At a Treg / DC ratio of 5:1, anti-DNP CAR Tregs, but not anti-CD19 CAR Tregs, further reduced the expression of CD86 and CD80 on DCs (FIGS. 4G-4H, 14A-14B). These experiments demonstrated that anti-DNP CAR Tregs, upon stimulation by DNP-labeled cells, can suppress the activation status of DCs. Example 7: Anti-DNP CAR Tregs can be stimulated by transplants in vivo It was next studied whether anti-DNP CAR Tregs can be stimulated by DNP-labeled transplants in vivo. Dorsal skin was harvested from Balb / c mice, injected with ManDNP-6, and transplanted to the recipient immunocompetent C57BL / 6 mice on day 0. Anti-DNP CAR Tregs were then intravenously injected on days 2, 3, and 4, followed by the collection of transplants for immune cell analysis on day 7 (FIG.5A). Compared to control mice, mice receiving the intravenous injections of anti-DNP CAR Tregs showed the presence of HA+CD25+CD4+cells in the skin allograft (FIGS.5B-5C), indicating the successful migration of anti-DNP CAR Tregs to the transplant site. Compared to untreated skin allografts, skin preloaded with ManDNP-6 resulted in a significantly higher fraction of HA+CD25+Tregs among CD4+cells (FIGS.5B-5C) and a much higher percentage of HA+cells among Tregs in the allograft (FIG.5D), substantiating the in situ stimulation and expansion of anti-DNP CAR Tregs by DNP-labeled transplants. The numbers of CD4+and CD8+T cells in the skin allograft was also analyzed. Adoptive transfer of anti-DNP CAR Tregs was able to increase the fraction of CD4+cells while exerting negligible effects on the fraction of CD8+cells (FIGS.5E-5F). With the pre-loading of ManDNP-6 into skin allografts, adoptively transferred anti-DNP CAR Tregs further increased the number of CD4+T cells (FIGS.5E-5F) and CD4+ / CD8+ratio (FIG.5G) in the skin allografts. These experiments demonstrated that ManDNP-6 loaded skin allografts can stimulate adoptively transferred anti-DNP CAR Tregs in situ, and thus generate an immunosuppressive tissue environment. Example 8: Anti-DNP CAR Tregs prolong the survival of skin allograft After demonstrating that anti-DNP CAR Tregs can suppress effector CD8+T cells and DCs in a DNP-specific manner and become stimulated by DNP-labeled cells in vitro and in7950-111351-02vivo, their therapeutic efficacy in a mouse skin allotransplantation model was next studied. Full-thickness skin was harvested from Balb / c mice, injected with ManDNP-6 or PBS at multiple sites, and transplanted to histocompatibility complex (MHC) mismatched, immunocompetent C57BL / 6 mice (FIG.5H) (Cheng et al., J Vis Exp, 119:e55105, 2017; Eskandari et al., Sci Transl Med 12:eaaw4744, 2020; Markees et al., J Clin Invest 101:2446- 2455, 1998). Anti-DNP CAR Tregs with C57BL / 6 background were intravenously injected once daily for three consecutive days (days 2-4). Mice without CAR Treg treatment exhibited rapid immune rejection of skin allografts, starting from day 3 post-transplantation (FIGS.5I- 5J). While mice receiving PBS-treated skin allograft and anti-DNP CAR Tregs showed delayed allograft rejection compared to mice without CAR Treg treatment, the overall survival time of skin allografts failed to show any improvement (FIGS.5I-5J). In contrast, mice receiving ManDNP-6-loaded skin allograft and i.v. injection of anti-DNP CAR Tregs showed significantly improved allograft survival (FIGS.5I-5J). Histological analysis of skin allografts showed complete coagulation necrosis of the epidermis, exfoliated hair shafts, and fulminant neutrophilic infiltrates in mice without anti- DNP CAR Treg treatment (FIG.15A). Skin allografts in mice receiving PBS-treated skin allograft and anti-DNP CAR Tregs also exhibited multifocal ulceration of epidermis, loss of hair follicles, superficial serocellular crust, keratinocyte apoptosis and degeneration in epidermis, and infiltration of lymphocytes and macrophages (FIG.15B). In comparison, skin allografts from mice receiving ManDNP-6-treated skin allograft and anti-DNP CAR Tregs showed markedly attenuated allograft rejection and epidermal injury, as characterized by the irregularly thickened hyperplastic epidermis with retention of fibroadnexal apparatus and increased fibrous replacement in dermis, which suggest an attempt by the host to initiate tissue repair following the transplantation (FIG.15C). These experiments demonstrated the ability of anti-DNP CAR Tregs to alleviate the immune attack towards ManDNP-6 loaded skin allografts and prolong the overall survival of allografts. Discussion and Conclusion Transplantation has been long regarded as a promising treatment option to replace damaged cells, tissues, and organs, but is hampered by the lack of strategies to effectively control immune rejection without impairing the recipient’s immune system. Transplant- specific Tregs can potentially address these issues by confining the immunosuppressive activity to the transplantation site. However, the identification of endogenous transplant-7950-111351-02specific antigens for the development of transplant-specific Tregs remains a challenge. Even more challenging is the development of universal transplant-specific Treg therapy that can be widely applicable to different types of transplants. Disclosed herein is a strategy to develop transplant-specific CAR Tregs, by manually installing unique chemical tags (e.g., DNP) onto the membrane of transplanted cells and designing CAR Tregs that are specific to the artificial chemical tag. This approach can be universally applied to various types of transplants without having to customize the design of CAR Tregs for each type of transplant. In principle, the DNP-sugar and anti-DNP CAR Tregs could be used as an off-the-shelf product for transplantation applications. It is demonstrated herein that ManDNP-6 can metabolically label various types of cancerous and non-cancerous cells with DNP groups, and that DNP-labeled cells specifically stimulate anti-DNP CAR Tregs, as evidenced by the upregulated expression of Helios and CD69 activation markers. Anti-DNP CAR Tregs, upon stimulation by DNP-labeled cells, inhibit the proliferation of effector CD8+T cells and downregulate the expression of activation markers on DCs. This DNP-specific stimulation of anti-DNP CAR Tregs is important for achieving transplant-specific protection without causing systemic non-specific immune suppression. By preloading ManDNP-6 into skin allografts, it is shown herein that adoptively transferred anti-DNP CAR Tregs migrate to the transplantation site, become stimulated in situ, mitigate immune rejection, and prolong the survival of allografts. In addition to DNP, other chemical tags are contemplated for the design of transplant- specific CAR Tregs. Suitable chemical tags include those with sufficient metabolic labeling efficiency and for which there are high-affinity antibodies available to extract scFv for CAR design. DNP is a relatively small functional group so that DNP-sugars can still undergo the metabolic glycoengineering processes and be expressed on the cell membrane. DNP is also large enough to induce high-affinity antibodies that can be used to extract the scFv for the design of anti-DNP CAR. In summary, the present disclosure describes the development of a facile and universal strategy to develop transplant-specific Tregs for the immune protection of transplants. First, DNP-sugars that can metabolically label various types of cells (including human primary cells) with DNP groups were developed. With the design and synthesis of an anti-DNP CAR construct, anti-DNP CAR Tregs were generated that can specifically recognize both DNP-bearing molecules and DNP-labeled cells. Upon the recognition of DNP ligands, anti-DNP CAR Tregs are stimulated and enable the suppression of effector CD8+T cells and DCs. It is further shown herein that anti-DNP CAR Tregs can be stimulated by7950-111351-02DNP-tagged transplants in vivo and improve the survival of skin allografts in a murine skin transplantation model. DNP-sugars and anti-DNP CAR Tregs can be universally applied to various types of transplants. The disclosed CAR Treg system provides a new avenue to achieve transplant-specific immunosuppression, a long-standing goal for tissue and organ transplantation. It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

7950-111351-02CLAIMS 1. A method of reducing allograft rejection or risk of allograft rejection in a subject, comprising: metabolically labeling an allograft tissue or organ with a chemical tag to produce a labelled allograft tissue or organ; transplanting the labelled allograft tissue or organ in the subject; and administering to the subject a therapeutically effective amount of T regulatory (Treg) cells expressing a chimeric antigen receptor (CAR) that specifically binds the chemical tag displayed on the allograft tissue or organ, thereby reducing the allograft rejection or the risk of allograft rejection in the subject.

2. The method of claim 1, wherein the chemical tag comprises dinitrophenyl (DNP) or a DNP analog, fluorescein or a fluorescein derivative, biotin or a biotin derivative, sulfanilamide or a sulfanilamide derivative, a nitrated amino acid, a modified nucleotide or nucleoside, a biocompatible polymer, or an oxidized lipid.

3. The method of claim 2, wherein: the DNP analog comprises dinitrochlorobenzene (DNCB), 3-nitro-4- hydroxyphenylacetate (NHPA), 2,4-dinitrophenol, picric acid, 2,5-dinitrophenol, m- nitrophenol, 2,6-dinotrophenol, or p-nitroaniline; the nitrated amino acid comprises nitrotyrosine; the modified nucleotide or nucleoside comprises 5-bromo-2'-deoxyuridine (BrdU) or 5-methylcytidine; the biocompatible polymer comprises polyethylene glycol (PEG) or oligo(ethylene glycol) (OEG); or the oxidized lipid comprises 4-hydroxynonenal.

4. The method of claim 2, wherein the chemical tag comprises DNP.

5. The method of claim 4, wherein metabolically labeling the allograft tissue or organ with DNP comprises contacting the allograft tissue or organ with a DNP sugar.

6. The method of claim 5, wherein the DNP sugar comprises N-(2,4- dinitrophenylamino)-acetyl mannosamine (ManDNP-2), tetraacetyl-N-(2,4-7950-111351-02dinitrophenylamino) acetyl mannosamine (Ac4ManDNP-2), N-(2,4-dinitrophenylamino)- caprylic mannosamine (ManDNP-6), tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic mannosamine (Ac4ManDNP-6), N-(2,4-dinitrophenylamino)-acetyl galactosamine (GalDNP- 2), tetraacetyl-N-(2,4-dinitrophenylamino) acetyl galactosamine (Ac4Gal-DNP-2), N-(2,4- dinitrophenylamino)-caprylic galactosamine (GalDNP-6), tetraacetyl-N-(2,4- dinitrophenylamino)-caprylic galactosamine (Ac4Gal-DNP-6), N-(2,4-dinitrophenylamino)- acetyl neuraminic acid (Neu5DNP-2), tetraacetyl-N-(2,4-dinitrophenylamino)-acetyl neuraminic acid (Ac4Neu5DNP-2), N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Neu5DNP-6), or tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Ac4Neu5DNP-6).

7. The method of claim 5, wherein contacting the allograft tissue or organ with the DNP sugar comprises immersing the allograft tissue or organ in a solution containing the DNP sugar or perfusing the allograft tissue or organ with a solution containing the DNP sugar.

8. The method of claim 5, wherein contacting the allograft tissue or organ with the DNP sugar comprises injecting the allograft tissue or organ with the DNP sugar.

9. The method of claim 1, wherein the Treg cells are autologous Treg cells.

10. The method of claim 1, wherein the Treg cells are allogeneic Treg cells or Treg cells obtained from universal stem cells.

11. The method of claim 1, wherein the CAR comprises: a monoclonal antibody or antigen-binding fragment thereof that specifically binds the chemical tag; a hinge region; a transmembrane domain; and one or more intracellular signaling domains.

12. The method of claim 11, wherein the antigen binding fragment is a single chain variable fragment (scFv).7950-111351-0213. The method of claim 12, wherein the scFv comprises, in the N-terminal to C- terminal direction, a variable light (VL) domain, a peptide linker, and a variable heavy (VH) domain.

14. The method of claim 11, wherein: the hinge region comprises a CD8 hinge region; the transmembrane domain comprises a CD8 transmembrane domain; and / or the one or more intracellular signaling domains comprise a CD28 signaling domain and a CD3ζ signaling domain.

15. The method of claim 1, wherein the Treg cells expressing the chemical tag- specific CAR are produced by transducing the Treg cells with a lentivirus vector encoding the CAR.

16. The method of claim 1, wherein the CAR-expressing Treg cells are administered to the subject 1 to 5 days following transplant.

17. The method of claim 16, wherein the subject is administered at least 1, at least 2, at least 3, at least 4 or at least 5 doses of the CAR-expressing Treg cells.

18. The method of claim 1, wherein the allograft tissue or organ comprises skin, blood vessel, bone, bone marrow, stem cells, cartilage, tendon, ligament, nerves, cornea, heart valve, heart, intestine, stomach, kidney, liver, lung, pancreas, or uterus.

19. A kit, comprising: a chemical tag, wherein the chemical tag comprises dinitrophenyl (DNP) or a DNP analog, fluorescein or a fluorescein derivative, biotin or a biotin derivative, sulfanilamide or a sulfanilamide derivative, a nitrated amino acid, a modified nucleotide or nucleoside, a biocompatible polymer, or an oxidized lipid; a viral vector encoding a chimeric antigen receptor (CAR) that specifically binds the chemical tag; engineered CAR T regulatory (Treg) cells specific for the chemical tag; and / or7950-111351-02instructions, a Treg isolation kit, cell culture media, cell culture flasks, tubes, syringes, needles, or any combination thereof.

20. The kit of claim 19, wherein the chemical tag comprises DNP in the form of a DNP sugar.

21. The kit of claim 20, wherein the DNP sugar comprises N-(2,4- dinitrophenylamino)-acetyl mannosamine (ManDNP-2), tetraacetyl-N-(2,4- dinitrophenylamino) acetyl mannosamine (Ac4ManDNP-2), N-(2,4-dinitrophenylamino)- caprylic mannosamine (ManDNP-6), tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic mannosamine (Ac4ManDNP-6), N-(2,4-dinitrophenylamino)-acetyl galactosamine (GalDNP- 2), tetraacetyl-N-(2,4-dinitrophenylamino) acetyl galactosamine (Ac4Gal-DNP-2), N-(2,4- dinitrophenylamino)-caprylic galactosamine (GalDNP-6), tetraacetyl-N-(2,4- dinitrophenylamino)-caprylic galactosamine (Ac4Gal-DNP-6), N-(2,4-dinitrophenylamino)- acetyl neuraminic acid (Neu5DNP-2), tetraacetyl-N-(2,4-dinitrophenylamino)-acetyl neuraminic acid (Ac4Neu5DNP-2), N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Neu5DNP-6), or tetraacetyl-N-(2,4-dinitrophenylamino)-caprylic neuraminic acid (Ac4Neu5DNP-6).

22. The kit of claim 19, wherein the viral vector is a lentiviral vector.

23. The kit of claim 19, wherein the CAR comprises: a monoclonal antibody or antigen-binding fragment thereof that specifically binds the chemical tag; a hinge region; a transmembrane domain; and one or more intracellular signaling domains.

24. The kit of claim 23, wherein: the antigen binding fragment is a single chain variable fragment (scFv); the hinge region comprises a CD8 hinge region; the transmembrane domain comprises a CD8 transmembrane domain; and / or the one or more intracellular signaling domains comprise a CD28 signaling domain and a CD3ζ signaling domain.7950-111351-0225. The kit of claim 19, wherein the engineered CAR Treg cells are obtained from stem cells or an allogeneic source.

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