Gene editing to induce FOXP3 expression in human CD4⁺ T cells leads to a stable regulatory phenotype and function

A gene-editing strategy to bypass epigenetic silencing in human peripheral blood CD4⁺ T cells

Transcription of FOXP3 in conventional T cells is epigenetically silenced via methylation events occurring within the T_reg-specific demethylated region (TSDR) in intron 1 of FOXP3 (24). We hypothesized that insertion of a transcriptional promoter downstream of the TSDR, but upstream of the protein start codon, could drive constitutive FOXP3 expression in conventional T cells. We have previously developed methodologies to insert novel sequences into genetic loci of interest by HDR using engineered nucleases and adeno-associated virus (AAV) donor templates (25–29). We therefore designed a gene-editing strategy to insert an MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) promoter into the FOXP3 locus to drive expression of endogenous FOXP3 in human T cells (Fig. 1A). For ease of detection of edited cells by flow cytometry, our initial editing strategy also introduced a coding sequence for green fluorescent protein (GFP) that upon successful HDR would become in frame with the N terminus of endogenous FOXP3 coding sequence. This mimics the sequence of Foxp3^tm2Ayr mice (30), which have normal T_reg development. Our gene-editing strategy should thus result in MND-regulated transcription of the endogenous FOXP3 gene, encoding an N-terminal GFP-FOXP3 fusion protein.

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We designed transcription activator-like effector nuclease (TALEN) pairs (sense and antisense) to induce DNA double-strand breaks near the FOXP3 start codon (fig. S1A). The donor DNA template was delivered using a recombinant AAV6 vector containing FOXP3 homology regions 5′ and 3′ of the TALEN-cleavage site flanking an MND promoter and GFP coding sequence (Fig. 1A). After purification and activation using CD3/CD28 beads, primary human CD4⁺ T cells were electroporated with forward and reverse FOXP3-specific TALEN mRNA pairs and subsequently cultured in medium containing the AAV6 donor template (Fig. 1B). To serve as a negative control, a portion of the activated CD4⁺ T cells were “mock”-edited by electroporating without TALEN mRNA and then culturing in medium without AAV. In genomic DNA (gDNA) extracted from CD4⁺ T cells treated with TALEN mRNAs only, we found an 80% frequency of DNA insertions or deletions (indels) at the target site, indicating efficient on-target cutting (fig. S1B). We did not detect any indels within any of the 15 highest-scoring possible off-target sites (table S1), suggesting target-specific cleavage using these TALENs. Control cells treated with the AAV donor template alone did not express detectable GFP at any time after transduction. Cells that received both FOXP3-specific TALEN mRNA and AAV expressed GFP, and GFP expression correlated linearly with that of intracellular FOXP3 (Fig. 1C), consistent with successful HDR driving expression of the FOXP3 gene with an N-terminal GFP fusion. On average, our editing protocol resulted in 37% GFP⁺ cells when cells were treated with both TALEN mRNA and the AAV donor template (Fig. 1D). To verify successful editing at the molecular level, GFP⁺ cells were purified by fluorescence-activated cell sorting (FACS) and gDNA amplified by polymerase chain reaction (PCR) using FOXP3-specific primers outside of both homology arms of AAV donor template (Fig. 1E). Only gDNA from cells treated with both TALEN and AAV gave the expected increase in amplicon size due to insertion of donor template (Fig. 1E). In the sample treated with TALEN and AAV, gDNA was isolated from FACS-purified GFP⁺ cells. Hence, most amplicons contain MND-GFP sequences. The two faint bands in the sample are nonspecific amplification products from the PCR. In addition, sequence analysis of GFP-sorted gene-edited cells confirmed the presence of the donor template integrated seamlessly at the FOXP3 locus (fig. S1C). These results validated our HDR approach as a method to induce high expression of the FOXP3 transcription factor by seamless, targeted gene editing in human CD4⁺ T cells.

FOXP3 editing promotes expression of proteins that discriminate thymic-derived T_reg from T_eff

The expression of FOXP3 is critical in establishing and maintaining the T_reg transcriptome. We first compared the phenotype of gene-edited T cells expressing FOXP3 driven by the inserted MND promoter (referred to hereafter as edT_reg) with that of tT_reg or T_eff isolated from the same peripheral blood sample. tT_reg and T_eff were purified by FACS based on the following cell surface marker staining: CD4⁺CD25⁺⁺CD127⁻ (tT_reg) and CD4⁺CD25⁻ (T_eff). Example flow plots from before and after sorting are shown in fig. S2. T_eff, tT_reg, GFP⁺ edT_reg, and mock-edited cells were expanded in parallel by culturing with CD3/CD28 beads followed by immunostaining for intracellular and surface markers associated with tT_reg (Fig. 2A). Both tT_reg and GFP⁺ edT_reg expressed similarly high amounts of FOXP3 and CD25 (interleukin-2 receptor α or IL-2Rα) and reduced CD127 (IL-7Rα) (Fig. 2, B and C). Like tT_reg, edT_reg up-regulated expression of CTLA-4 and ICOS (Fig. 2, B and C). It is known that expression of FOXP3 and other T_reg markers is transiently altered in T_eff after activation (31); accordingly, both T_eff and mock-edited cells showed moderate expression of T_reg-associated markers (such as CD25 and FOXP3 up-regulation) in response to cell activation. After bead removal and subsequent cell culture, edT_reg retained high expression of FOXP3, CD25, CTLA-4, and ICOS, whereas mock-edited cells diminished expression of T_reg-associated markers. edT_reg expressed intermediate quantities of the transcription factor Helios relative to tT_reg or mock-edited T cells. At the same time in culture, we also compared expression of intracellular cytokines interferon-γ (IFN-γ), IL-2, tumor necrosis factor–α (TNF-α), and IL-4 after a 5-hour stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Fig. 2D). Expression profiles of IFN-γ, IL-2, TNF-α, and IL-4 (Fig. 2D) were similarly reduced in tT_reg and edT_reg compared to mock-edited cells and T_effs. To assess whether CD25 up-regulation was correlated with increased downstream signaling, we measured the phosphorylation of signal transducer and activator of transcription 5 (STAT5) in response to varying IL-2 concentrations. edT_reg had an increased sensitivity to low doses of IL-2 in comparison to mock-edited cells (fig. S3).

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We next performed a global comparison of the transcriptomes of edT_reg, tT_reg, and T_eff populations isolated from four different human donors using RNA sequencing (RNAseq). RNA was collected directly from FACS-purified cells after a 48-hour stimulation using CD3/CD28 beads and a subsequent 48-hour rest period (without bead stimulation; detailed experimental timeline is shown in fig. S4). Using rlog-transformed count values on a subset of genes showing the highest degree of global expression variation, we performed hierarchical clustering of the samples. We also looked at the deviation from the gene’s average expression across all samples and plotted this as a heat map, with samples showing the highest degree of similarity in gene expression being nearest to each other. Figure S5 shows the 100 most variable genes between each T cell subset. We observed the highest degree of clustering between the same T cell subset (T_eff, tT_reg, or edT_reg). The next highest degree of similarity was observed between the tT_reg and edT_reg subsets. This hierarchy was maintained when we performed the clustering based on 30 transcripts previously shown to distinguish tT_reg from T_eff regardless of activation status (Fig. 2E) (32). In summary, edT_regs differ from mock-edited T cells in that they exhibit phenotypic characteristics of tT_reg, including expression of canonical surface and intracellular markers of tT_reg, a tT_reg-like cytokine production profile, greater sensitivity to IL-2, and a post-stimulation gene signature that is similar to sorted tT_reg derived from the same donor.

In vitro immunosuppression by edT_reg is dependent on functional FOXP3

A defining characteristic of tT_reg is their ability to suppress the activation of autologous T_eff. We first tested whether edT_reg could suppress the proliferation of CD4⁺ T cells after polyclonal stimulation in vitro. We sorted edited CD4⁺ T cells based on GFP expression. GFP⁺ versus GFP⁻ sorted fractions were cocultured with fluorescently labeled autologous CD4⁺ T_eff undergoing CD3/CD28 bead stimulation for 96 hours. Proliferation was read out using flow cytometry based on dilution of the fluorescent dye. Only the GFP⁺ cells suppressed the proliferation of T_eff, showing that edT_reg function is linked to the FOXP3⁺/GFP⁺ phenotype (Fig. 3A).

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Because our editing strategy up-regulates expression of the endogenous FOXP3 transcript by knocking in a strong upstream promoter, edT_regs generated using input CD4⁺ T cells isolated from patients with IPEX, with loss-of-function mutations in FOXP3, are expected to be nonsuppressive. To directly demonstrate that edT_reg immunosuppression is based on activation of endogenous expression of a functional FOXP3 protein, we compared the ability of editing to confer a suppressive phenotype between a healthy control donor and two patients with IPEX with FOXP3 mutations p.I363V (IPEX1) and p.A80Dfs*49 (IPEX2). Edited CD4⁺ T cells from the healthy subject gained suppressive function, whereas those from the patients with IPEX did not (Fig. 3B). Note that edT_reg derived from CD4⁺ T cells isolated from IPEX subjects expressed similar amounts of GFP-FOXP3 fusion protein compared with edT_reg derived from the healthy control donor (Fig. 3C). These results demonstrate that edT_reg from healthy donors can suppress expansion of activated T_eff in vitro, and this property is driven by increased expression of the functional endogenous FOXP3 in edited cells.

Comparison of edT_reg with T cells expressing lentiviral-delivered FOXP3

Lentiviral (LV) transfer of a FOXP3 cDNA expression cassette into conventional T cells has been shown to confer a T_reg-like phenotype and suppressive characteristics in vitro and in vivo (21, 33). As a comparison for our editing strategy, we generated an LV construct to deliver a cDNA encoding the same GFP-FOXP3 fusion protein made by our edT_reg (Fig. 4A). The FOXP3 gene is located on the X chromosome, and because our experiments were conducted in CD4⁺ cells derived from male donors, there is only one FOXP3 allele per cell. The gene-editing and viral transduction procedures produced similar proportions of GFP⁺FOXP3⁺ cells (Fig. 4A). In contrast to the single copy of donor DNA per cell in edT_reg, LV-treated cells (LV T_reg) had an average of 3.0 LV integrations per GFP⁺ cell genome. Despite their copy number differences, the GFP mean fluorescence intensity (MFI) of GFP⁺ cells was consistently lower in LV T_reg than in edT_reg (Fig. 4B), perhaps indicating more efficient expression from a genomic versus cDNA context or LV integration in transcriptionally less permissive loci. FACS-purified LV T_reg cells lost GFP expression more rapidly during 5 weeks in culture than edT_reg (Fig. 4C; starting % GFP⁺ > 99% for both). When analyzed approximately 2 weeks after sorting, both methods of enforcing FOXP3 expression skewed the T_eff toward tT_reg phenotypes, including up-regulation of CD25, CTLA-4, and Helios and down-regulation of IL-2, TNF-α, and IFN-γ (Fig. 4D). Except for FOXP3, the percentage of cells expressing T_reg markers, as well as the mean expression (as assessed by MFI) per cell, were similar between edT_reg and LV T_reg. We also demonstrated increased expression of the complement binding protein CD59 (based on flow cytometry staining; Fig. 4D), further validating our RNAseq data derived from tT_reg and edT_reg. Functionally, edT_reg and LV T_reg were equivalent in their ability to suppress polyclonal T_eff proliferation in vitro (Fig. 4E).

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Enrichment and expansion of edT_reg using methods relevant for cell product manufacturing

T_reg cells have a growth disadvantage in vitro and can be outcompeted by T_eff (34). To obtain edT_reg in numbers and purity sufficient for a therapeutic application, it will be necessary to expand edT_reg in vitro and useful to enrich for successfully edited cells. After FACS-purifying GFP⁺ edT_regs 2 days after editing (as in Fig. 2A), cells were cultured for 7 or 14 days. The edT_reg expanded 23- to 48-fold in 7 or 14 days, respectively, yielding an average of 78 × 10⁶ cells at 14 days from 1.5 × 10⁶ sorted cells (Fig. 5A). As expected, greater expansion was achieved for mock-edited cells (158-fold). After 14 days of expansion, T cells from both mock-edited and edT_reg groups exhibited a diverse TCR repertoire by Vβ spectratype analysis (fig. S6). We also assessed the methylation status of the TSDR CpGs within these cells. The TSDR affects transcription of the upstream FOXP3 promoter using epigenetic mechanisms. A TSDR with mostly nonmethylated CpGs is permissive for FOXP3 expression and is an epigenetic signature of tT_reg, whereas CpGs in the TSDR of T_eff are highly methylated, largely silencing FOXP3 expression. The MND promoter of edT_reg is inserted 2.1 kb downstream of the TSDR, and its ability to drive FOXP3 expression in targeted cells could reflect demethylation of the TSDR or the ability to drive transcription despite TSDR methylation. Bisulfite conversion and subsequent colony sequencing of gDNA extracted from expanded edT_reg and mock-edited cells revealed that nearly all CpGs within the TSDR of expanded edT_reg were methylated (97%), as were those of mock-edited T cells (94%), whereas the TSDR CpGs of tT_reg were minimally methylated (2.4%; Fig. 5B). These results imply that the MND promoter integrated downstream of TSDR bypasses the epigenetic silencing mediated by the TSDR at the endogenous upstream promoter. In addition, neither the presence of the MND promoter nor up-regulation of FOXP3 expression altered the TSDR methylation status.

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To allow enrichment of successfully edited cells, we next designed donor templates replacing the GFP coding sequence with that of a surface tag for use in cell selection. We tested two clinically relevant approaches for affinity selection: a truncated human low-affinity nerve growth factor receptor (LNGFRt; Fig. 5C) (35) and a truncated human epidermal growth factor receptor (EGFRt; fig. S7B) (36) that are expressed on the cell surface but lack intracellular signaling capabilities. Both epitope tags were in frame with a 2A ribosome-skip peptide (37) and the FOXP3 coding sequences. HDR resulted in surface expression of LNGFRt or EGFRt (linked at the C terminus to most of the 2A peptide) and endogenous FOXP3 (with one additional proline at the N terminus from the 2A peptide). After editing primary CD4⁺ T cells using the relevant AAV6 donor template, cell surface expression of each epitope was detected, allowing enrichment of edited cells to >80% purity using antibody affinity beads (fig. S7, A and B). LNGFRt-enriched (Fig. 5D) or EGFRt-enriched (fig. S7C, left) edT_reg expressed FOXP3 at high MFIs and had tT_reg-like phenotypes such as CD25⁺⁺CD127^−/low and reduced intracellular inflammatory cytokines (Fig. 5E and fig. S7C, right). Next, we tested the in vitro suppressive function of LNGFRt-enriched edT_reg compared with mock-edited T cells or purified tT_reg (Fig. 5, F and G). LNGFRt⁺ edT_reg exhibited suppressive function equivalent to tT_reg at all ratios of T_reg:T_eff. Thus, edT_reg can be generated using a strategy that introduces a clinically relevant selection marker and can be expanded ex vivo to large numbers for potential clinical application. Enriched cells maintain a distinct immunophenotype that is independent of TSDR methylation status even after expansion ex vivo.

Adoptive transfer of autologous human edT_reg suppress T_eff-mediated GvHD in NSG mice

We next tested whether edT_reg could suppress an immune response in vivo using a xenogeneic graft-versus-host disease (xeno-GvHD) model. In this model, injection of human T_effs into minimally irradiated immunodeficient nonobese diabetic (NOD)–scid–IL2rγ^NULL (NSG) mice provokes a polyclonal, major histocompatibility complex (MHC)–dependent immune response against multiple host tissues (38) and leads to symptoms of GvHD including hair loss, skin lesions, and weight loss. We first determined whether edT_reg alone could become pathogenic in this model. We used AAV FOXP3[MND.GFPki] as the donor template in these experiments for ease of identifying edited cells in postmortem mouse tissues. After editing primary human CD4⁺ T cells, GFP⁺ cells were obtained by FACS and expanded for 7 days; mock-edited cells were not FACS-purified before expansion. Next, 8 × 10⁶ edT_reg or mock-edited T cells, or 4 × 10⁶ CD4⁺ T_effs from the same human donor were injected into separate irradiated NSG mice and monitored for symptoms of GvHD several times weekly. All the mice treated with T_eff developed overt symptoms of GvHD and were euthanized at predetermined humane endpoints as early as day 12 (fig. S8). GvHD was delayed in mice receiving mock-edited cells, with most reaching humane endpoints before 50 days after cell injection. In contrast, none of the mice receiving edT_reg alone showed symptoms of GvHD and all survived through the 50-day experimental endpoint (fig. S8). Histological sections of tissues obtained from the T_eff cohort showed extensive lymphocyte infiltration into multiple tissues including the lung, liver, intestine, skin, kidney, and pancreas (fig. S9) and higher histological scores relative to mice that received no T cells, confirming a diagnosis of GvHD.

To determine whether edT_reg could suppress the development of GvHD in this model, 8 × 10⁶ edT_reg, mock-edited T cells, or tT_reg (CD4⁺CD25⁺⁺CD127⁻ cells purified by FACS and expanded in culture) from the same peripheral blood sample were injected into NSG mice immediately after irradiation and allowed to engraft for 3 days before injection of 4 × 10⁶ autologous CD4⁺ T_eff (2:1 T_reg:T_eff ratio; Fig. 6A). A subset of animals from each cohort was sacrificed 14 days after injection to assess the effect of T_reg on T_eff expansion before the onset of overt GvHD; all remaining animals were monitored for up to 50 days, before being euthanized for tissue analyses. Animals engrafted with either edT_reg or tT_reg delayed severe weight loss (fig. S10A), showed lower disease score (fig. S10B), and had significantly (P = 0.0003) improved survival rates relative to recipients of mock-edited T cells (Fig. 6B).

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On day 14 after adoptive transfer of T_eff, both the percentage and absolute number of human CD4⁺ T cells were decreased in the blood, spleen, liver, and lung of mice co-engrafted with edT_reg or tT_reg (Fig. 6C). edT_regs (GFP⁺CD4⁺) were present in all examined tissues of edT_reg recipient mice (Fig. 6D), ranging from a mean of 5% of CD4⁺ T cells in bone marrow (BM) to 28% in peripheral blood. In addition, GFP⁻CD4⁺T cells (which were mostly T_eff) in edT_reg or tT_reg recipients exhibited a more naïve (CD45RO⁻CD62L⁺) phenotype and reduced expression of activation markers (CD38⁺ or CD40L⁺) compared with CD4⁺ T cells in mock-edited or T_eff only groups (fig. S11A). Compared with mock-edited and T_eff only groups, GFP⁻CD4⁺ T cells in the peripheral blood of edT_reg-engrafted cohorts reduced expression of intracellular proinflammatory cytokines (IFN-γ and IL-17A) and recipients of either edT_reg or tT_reg exhibited a lower T helper 1 (T_H1)/T_H2 ratio in the lung (fig. S11B). These data suggest that edT_regs behave similarly to tT_reg cells in this model, trafficking to multiple tissues and suppressing the activation of autologous CD4⁺ T cells.

GFP-expressing cells remained detectable in tissues (ranging from mean of 9% of CD4⁺ T cells in liver to 27% in lung) of animals engrafted with edT_reg evaluated at the 50-day time point, demonstrating a stable phenotype despite a highly inflammatory environment (Fig. 6D). In most samples, GFP⁺ cells retained expression of T_reg markers including increased FOXP3, CD25, and CTLA-4 and reduced CD127 (Fig. 6E). In some tissue samples (e.g., day 14 lung and day 50 blood and spleen), a proportion of GFP⁺ lymphocytes expressed CD127, consistent with a previous finding for in vivo activated T_reg (39). FOXP3 was present at a high MFI in GFP⁺ cells in the spleen on day 50 as shown by intracellular staining; however, FOXP3 expression was difficult to quantify by our intracellular staining procedure in samples with low numbers of GFP⁺ cells in other organs. Importantly, and consistent with a regulatory phenotype, FOXP3⁺GFP⁺ cells on day 50 after transfer had low intracellular IL-2 compared with GFP⁻ cells (Fig. 6F). Together, these findings demonstrate sustained engraftment and functional regulatory activity of edT_reg in a robust MHC class II–dependent in vivo inflammatory model.

To determine whether engineered CD4⁺ T cells with analogous T_reg-like features and function could be developed using an alternative designer nuclease platform and a similar HDR-editing strategy, we developed a CRISPR-Cas9 reagent targeting FOXP3. We established a guide RNA (gRNA) targeting the first coding exon of human FOXP3 (upstream of the TALEN cleavage site) and an AAV HDR-donor construct with homology arms immediately upstream and downstream of Cas9-targeted sequences as shown schematically in fig. S12A. Cas9/gRNA cleaved the FOXP3 target at a high efficiency (88.7%; fig. S12B, left) with limited, in silico predicted, off-target site activity (fig. S12B, right, and table S2). Using the timeline described in fig. S12C, CRISPR-Cas9–mediated gene editing, LNGFR affinity enrichment, and cell expansion were performed to generate LNGFRt⁺ edT_reg. Figure S12D shows an average HDR rate of 41% for generation of LNGFRt⁺ edT_reg using independent donors. After affinity enrichment and subsequent expansion, LNGFRt⁺ edT_reg exhibited high purity with ~80-fold expansion (fig. S12E). Comparable to TALEN-generated edT_reg, Cas9-edited LNGFRt⁺ edT_reg expressed T_reg markers and reduced the production of inflammatory cytokines including IL-2, TNF-α, and IFN-γ (fig. S12, F and G). Cas9-edited LNGFRt⁺ edT_reg also suppressed the xeno-GvHD immune response in NSG mice (fig. S12H). Because the edT_reg features observed after HDR-based gene editing were equivalent between the TALEN and CRISPR-Cas9 nuclease platforms, we conclude that enforcement of MND-driven FOXP3 expression is responsible for the observed phenotypes.

Antigen-specific mouse edT_regs limit priming of T_effs in a model of multiple sclerosis

For diseases where critical autoantigens are known (e.g., T1D or rheumatoid arthritis), antigen-specific T_reg may be more potent for controlling disease (40, 41). To investigate edT_reg function in an antigen-specific in vivo setting, we sought to edit T cells from myelin oligodendrocyte glycoprotein peptide fragment 35–55 (MOG)–specific TCR-transgenic mice [C57Bl/6-Tg(Tcra2D2,Tcrb2D2)1Kuch/J, abbreviated 2D2] (42). MOG challenge in combination with pertussis toxin disruption of the blood-brain barrier leads to experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Although tT_regs expand and traffic to lesions in the central nervous system (CNS), MOG-induced EAE is not controlled by endogenous antigen-specific tT_reg, possibly because of inflammatory T_eff cytokines (43). We hypothesized that adoptive transfer of antigen-specific 2D2 edT_reg could suppress T_eff expansion in the periphery before these activated effectors migrate to the CNS. To test this hypothesis, we designed TALEN and AAV donor template reagents that would mimic as closely as possible the editing strategy used to generate the human edT_reg (fig. S13A). After optimizing procedures for murine T cell stimulation and mRNA electroporation, we transfected murine T cells with mRNA encoding TALEN pairs specific for the first coding exon of mouse Foxp3. Seven to 9 days after transduction, ~80% of alleles contained indels based on colony sequencing of PCR-amplified gDNA (fig. S13B), indicating efficient target site cleavage. We next generated an AAV donor template that substituted mouse Foxp3 homology arms for the human sequences used in our previous HDR experiments; homology was proximal to, but not overlapping, the mouse TALEN binding sites. With slight modifications of the conditions used for human CD4⁺ T cell editing, including using AAV5 capsid for donor template transduction, we achieved ~25 to 30% editing rates (%GFP⁺) using 2D2 mCD4⁺ T cells isolated from spleen and lymph nodes (LNs) (Fig. 7A). GFP⁺ cells were phenotypically FOXP3⁺CD25⁺CTLA-4⁺ (Fig. 7B). CD4⁺ T cells were also isolated from 2D2^neg littermates (C57Bl/6) and edited, resulting in edT_reg with polyclonal TCR specificities for comparison with 2D2 edT_reg. To assess in vivo function, we adoptively transferred 3 × 10⁴ 2D2 CD4⁺ T_effs in combination with 3 × 10⁴ mock or edT_reg, respectively, into lymphopenic Rag1^−/− mice. Recipient mice were challenged with MOG_35–55 peptide in adjuvant followed by pertussis toxin to disrupt the blood-brain barrier. Figure 7C shows the experimental timeline of cell transfer, immunization, and cell analysis. In this model, recipient animals developed symptoms of EAE (drooping tail progressing to tail then hindlimb paralysis) beginning at ~7 to 10 days after cell transfer. The immune response to EAE induction in Rag1^−/− mice was robust, and the course of disease was not controlled by polyclonal or antigen-specific tT_reg or edT_reg (fig. S14), as predicted from previous studies (43).

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Although neither tT_reg nor edT_reg controlled EAE progression in this model, we assessed the effect of these immunosuppressive cells on the responses of T_effs within the LNs. Recipient mice were euthanized at day 7 after transfer, and inguinal and axillary LNs were collected. The percentage of LN CD45⁺CD4⁺ T cells were twofold lower in recipients of antigen-specific 2D2 edT_reg compared to recipients of mock-edited cells and 1.7-fold lower compared with recipients of polyclonal C57Bl/6 edT_reg. The absolute number of CD4⁺CD45⁺ T cells was markedly reduced in both edT_reg cohorts relative to the mock control, with 2D2 edT_reg and C57Bl/6 edT_reg having 49- and 18-fold fewer T cells, respectively. In all cohorts, most of the CD45⁺CD4⁺ cells were GFP⁻, and fewer GFP⁻ T cells from mice receiving 2D2 edT_reg expressed inflammatory cytokines IL-17A or IFN-γ (Fig. 7D). To determine whether the observed effect was due to reduced T_eff proliferation, some animals were injected with the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) before sacrifice and its incorporation into gDNA was detected after a “click” reaction by flow cytometry (Fig. 7E). We found that 2D2 edT_reg reduced the overall percentage of GFP⁻ cells that had incorporated EdU by 22 and 18% relative to groups treated with mock or polyclonal edT_reg, respectively. EdU incorporation was present in GFP⁺ cells from 2D2 edT_reg (~25%) and, to a lesser extent, from C57Bl/6 edT_reg (~10%) recipient cohorts, consistent with the ability of tT_reg to proliferate in vivo in response to self-antigen stimulation. These combined findings show that murine edT_regs function in vivo to restrain the priming phase of pathogenic T_eff and that antigen-specific edT_regs exhibit greater activity and expansion in comparison with polyclonal edT_reg.

Antigen-specific human T cells adopt a T_reg phenotype after FOXP3 editing and are immunosuppressive in vitro

Our findings from the murine EAE model suggested that antigen-specific edT_reg may have a greater efficacy in adoptive transfer applications than polyclonal edT_reg. To determine whether our editing approach would be feasible with antigen-specific human T cells, CD4⁺ T cells from HLA DRB1*0401 donors were initially expanded for 7 days in the presence of the following pooled influenza and tetanus antigens: Clostridium tetani toxin (TT), influenza virus matrix protein 1 (MP), and influenza virus hemagglutinin (HA). The cultures were subsequently activated with CD3/CD28 beads before gene editing (Fig. 8A). After editing, cells were further expanded in the antigen cocktail for 4 to 7 days. At this time, the average editing rate (GFP⁺) was 28 ± 2.1% (Fig. 8B). Antigen-specific cells were purified by FACS after labeling with a mixture of phycoerythrin (PE)–conjugated influenza and tetanus MHC-II tetramers. These tetramer-positive edT_reg (Tr⁺edT_reg) recapitulated the immunophenotype of activated tT_reg for canonical markers of T_regs: up-regulating expression of FOXP3, CD25, CTLA-4, LAG3, and Helios and suppressing IL-2 production (Fig. 8C and fig. S15A). Tr⁺edT_regs were able to suppress polyclonal activated autologous CD4⁺T_eff in vitro, unlike the Tr⁺Mock cells, indicating immunosuppressive function (fig. S15B).

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We next developed methods to assess the specificity of immunosuppression mediated by antigen-specific edT_regs. After edT_regs were generated according to the schema shown in Fig. 8D and sorted, GFP⁺ edT_regs were evaluated for antigen-specific immunosuppressive activity. Three days after editing, ~14% of cells were GFP⁺ and this population contained ~60% MP, HA, or TT Tr⁺ cells (Fig. 8D). FACS-sorted GFP⁺ cells were expanded for 10 days and subsequently cocultured with autologous T_eff (CD4⁺CD25⁻) and antigen-presenting cells [APCs; irradiated autologous peripheral blood mononuclear cells (PBMCs)], and either dimethyl sulfoxide (DMSO) (control stimulation) or a pool of MP, HA, and TT peptides (peptide stimulation). Strikingly, T_eff proliferation, measured by dye dilution, was inhibited only in the presence of both antigen-specific edT_reg and cognate peptides (Fig. 8, E and F), indicating the requirement for TCR engagement for edT_reg activity.

It is known that tT_regs suppress T_eff of the same TCR specificity, as well as those with different TCR specificities that are in physical proximity, termed bystander suppression (44). We performed studies to directly test the capacity of human edT_reg to suppress T_eff with differing antigen specificities. To do so, we produced T_eff with antigen specificities either identical to or different from the edT_reg (see fig. S16 for experimental schematics) as follows. CD4⁺ T cells were stimulated with MP peptide for 10 days. MP Tr⁺ T cells were then sorted; some were retained for use as MP-specific T_eff (MP-TCR T_eff), and the remaining Tr⁺ T cells were activated by CD3/CD28 beads and edited to integrate MND-LNGFR-2A into FOXP3 as described in Fig. 5 and fig. S12A. After 7 days of expansion, MP Tr⁺ LNGFR⁺ and LNGFR⁻ cells were sorted to obtain MP Tr⁺ edT_reg (MP edT_reg) and MP Tr⁺ mock-edited cells (MP Mock), respectively (fig. S16, A and B). Mock-edited cells were used to control for the number of T cells per well, which by themselves suppress proliferation rates of the responder cells due to competition for growth factors such as IL-2. As shown in fig. S16C, MP edT_reg, to a greater extent than MP mock-edited cells, suppressed MP-TCR-T_eff proliferation in response to polyclonal stimulation (CD3/CD28 beads), indicating polyclonal immunosuppression. We next tested suppression in response to cognate peptides based on cytokine production of cocultured T_eff, a more sensitive readout of immunosuppression where cell numbers were limited. Here, MP-peptide stimulated MP edT_reg suppressed production of TNF-α, IL-17A, and IL-2 by MP-TCR T_eff, whereas MP mock cells did not (fig. S16D). A similar strategy was used to test for bystander suppression of T_eff proliferation. HA-specific T_effs (HA-TCR T_effs) were made by transduction of autologous CD4⁺ with a lentivirus delivering an HA-specific TCR expression cassette (fig. S16E). Although both MP edT_reg and MP mock cells suppressed HA-TCR T_eff proliferation in the setting of polyclonal stimulation, fewer MP-TCR T_effs proliferated in the presence of MP edT_reg. MP edT_reg and MP mock cells similarly suppressed HA-TCR T_eff production of TNF-α, IFN-γ, and IL-2 with HA peptide stimulation, but MP edT_reg showed further suppression of the cytokines in the presence of both the MP and HA peptides (fig. S16G). These results imply that antigen-specific edT_regs require TCR engagement to inhibit production of inflammatory cytokines by T_effs and that edT_regs exhibit bystander suppressive activity on T_effs with a different peptide specificity when edT_regs encounter their cognate peptide. Together, these results demonstrate that CD4⁺ T cells from human peripheral blood can be enriched for target antigen specificity and modified by gene editing to impart tT_reg-like phenotypic and suppressive properties that retain antigen specificity.