Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T – B + NK – ). Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a complete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development, including the production of mature T cell populations with a broad T cell receptor (TCR) repertoire. Whole-genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies.

In this paper, we demonstrate that differentiation of JAK3-deficient human T cells is blocked at an early developmental stage. Similar to previous studies in mouse models, JAK3-deficient early human T cell progenitors undergo apoptosis at a high rate due to low expression of BCL2. We also demonstrate that correction of the human JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores the differentiation potential of early T cell progenitors. These corrected progenitors are capable of producing NK cells and mature T cell populations expressing a broad repertoire of T cell antigen receptors (TCRs). These studies establish a powerful system for determining the mechanism of immunodeficiency in human SCID patients and for testing pharmacological and genetic therapies for the disorder.

IPSC technology combined with in vitro differentiation systems also provides a powerful platform to recapitulate in vivo development. We and other groups have shown that OP9 stromal cells transduced with Notch ligand Delta-like-1 or Delta-like-4 (OP9-DL1/4) can contribute to a T cell inductive environment (). OP9-DL1/4 cells efficiently induce T lymphopoiesis from iPSC-derived CD34hematopoietic progenitor cells (HPCs), and these HPCs can be differentiated into functional CD8 T cells (). Despite the lack of proper major histocompatibility complex (MHC) expression on OP9 cells, the OP9-DL1/4 system has provided a valuable method to study early T cell commitment and thymocyte maturation in vitro ().

Allogeneic hematopoietic stem cell transplantation is currently the only established therapy for SCID; however, delayed immune recovery and graft-versus-host disease present significant risks (). Treatment by retroviral-based gene therapy has been successfully demonstrated for X-linked SCID () and ADA-SCID (). However, severe adverse effects of insertional mutagenesis have been observed with retroviral gene therapy (). Self-inactivating lentiviral vectors have been used effectively in recent clinical trials, but long-term follow-up is needed to thoroughly address safety concerns (). An alternative therapeutic strategy is one in which patient-specific induced pluripotent stem cells (iPSCs) are derived, and disease-causing mutations are corrected by gene targeting (). These corrected iPSCs could then be differentiated into hematopoietic progenitors for transplantation into patients to treat the disease (). The recent development of CRISPR/Cas9-enhanced gene targeting dramatically advances the practicality of this strategy ().

Severe combined immunodeficiency (SCID) describes patients with severe defects in T cells with or without accompanying defects in B cells. Naturally occurring mutations in the JAK3 gene (autosomal recessive) and the X-linked common gamma chain γgene (IL-2RG) are the most common Timmunodeficiencies. Gene ablation experiments in mice demonstrate that Jak3 is critical for early T cell differentiation and Jak3 knockout mice were found to have severely reduced numbers of mature B cells in the bone marrow and in the periphery (). The similarity of phenotypes of γc and JAK3 SCID suggests that the primary function of JAK3 is to transduce signals from γc-dependent cytokine receptors (IL-2R, IL-4R, IL-7R, IL-9R, IL-15RA, and IL-21R) to transcription factors (STATs) that activate downstream genes. Mice lacking Jak3 have a profound decrease in thymus cellularity. However, the residual thymocytes proceed to develop into mature T cells and reconstitute the peripheral population. In contrast, JAK3-deficient patients have few, if any, peripheral T cells (). To date, little is known about how JAK3 mutations affect human lymphocyte progenitor development.

To determine whether TCR rearrangement is reestablished in JAK3-corrected T cells, TCR Vβ typing was performed by flow cytometry and summarized in Figure 5 A. JAK3-corrected T cells expressed all 21 of the Vβ segments that we tested therefore, a broad TCR repertoire was restored. Finally, we examined the integrity of the TCR signaling pathway, a surrogate of T cell function, in JAK3-corrected T cells by measuring cell surface activation markers following anti-CD3/CD28 stimulation. On day 3 post-stimulation, the percentage of CD3CD25CD69T cells increased from 0.68% to 59.7% in JAK3-corrected T cells, similar to the increase observed in control cells (0.01%–37.6%) ( Figure 5 B). These data and results described above demonstrate that correction of the JAK3 C1837T (p.R613X) mutation by CRISPR/Cas9-enhanced gene targeting in an in vitro iPSC model system restores normal T cell development with the capacity to produce functional, mature T cell populations with a broad TCR repertoire.

To determine whether T cell development is restored after JAK3 gene correction, NK and T cell generation were verified, and T cell lineage commitment and maturation were analyzed. We and other groups have previously demonstrated in the OP9-DL4 in vitro system that T cell differentiation sequentially passes through intermediates observed in vivo: CD34CD7T/NK committed stage; CD7CD4CD8immature, SP stage; CD4CD8DP stage; and finally, CD3CD8TCRαβ mature stage. Mature T cells are polyclonal, proliferate, and secrete cytokines in response to mitogens (). Therefore, control, JAK3-deficient, and JAK3-corrected human iPSCs were differentiated into hematopoietic progenitors on OP9 monolayers, and CD34cells were positively selected with anti-CD34 magnetic beads. These cells were plated onto OP9-DL4 monolayers, and non-adherent cells were analyzed for lymphocyte markers at T cell induction day (TD) 14, 21, 28, and 35 ( Figure 4 ). In normal controls (green line), 1.2 × 10CD7cells (84% of cells counted in the lymphoid gate) were generated at T cell induction day 14 from 1–2 × 10CD34cells. At this stage, about 20% of CD7cells were CD7CD16CD56NK cells (2.4 × 10). T cell markers CD4, CD8, CD3, and TCR αβ were sequentially detected upon T cell maturation. As we reported previously, at T cell induction day 35 the NK population decreased (6.9 × 10), and more than 50% of residual cells were CD8 SP cells (1.2 × 10). In JAK3-deficient cells (blue line), only 4.5 × 10CD7cells (38.9% of cells counted in lymphoid gate) were generated at T cell induction day 14 from 1–2 × 10CD34cells. The number of total CD7cells decreased during extended culture and T cell markers CD3, CD4, CD8, and TCR αβ were not significantly expressed.

The 20-base CRISPR guide sequences were mapped to the human reference genome, allowing up to three mismatches in order to identify potential off-target sites ( Table 1 Figure S1 ). These sites were then analyzed for variations in the iPSC samples following CRISPR/Cas9-directed gene correction. Whole-genome sequencing of the one homozygous and two heterozygous corrected iPSC lines demonstrated that no mutations (SNVs nor indels) were introduced into the 1,450 potential off-target sites ( Table 2 ). These results demonstrate the specificity of CRISPR/Cas9-directed gene correction.

Whole-genome sequencing (WGS) was performed on three corrected iPSC clones and the uncorrected control (see Whole-Genome Sequencing and Analysis in Experimental Procedures ). Variants (from the reference genome) that were common to all four iPSC samples were excluded from further analysis. The remainder were screened to determine whether these variants were located in potential off-target sites. No variants were observed in potential off-target sites. BEDTools (v.2.17.0) () was used to search for non-excluded variants in potential off-target sites.

The potential for off-target, CRISPR/Cas9-directed genome modifications raises some concerns about the use of this approach for therapy in humans. In cancer cell lines, relatively high levels of off-target mutagenesis by Cas9-gRNAs have been described (). However, several groups have recently demonstrated by whole-genome sequencing (WGS) that off-target modifications are rare in human iPSCs and human embryonic stem cells (). To determine the specificity of CRISPR/Cas9-directed JAK3 correction in human SCID iPSCs, we performed WGS before and after gene correction. The genomes of two heterozygous and one homozygous corrected clones were sequenced. The two heterozygous clones were corrected with gRNA #2 + wild-type Cas9, and the homozygous clone was corrected with gRNA #1 + gRNA #2 + D10A nickase Cas9.

To determine whether normal T cell development can be restored in JAK3-deficient SCID patient cells, we corrected the JAK3 mutation in iPSCs by CRISPR/Cas9-enhanced gene targeting. Six guide RNAs within introns upstream and downstream of exon 14 were designed to target wild-type Cas9 or the D10A Cas9 nickase near the C1837T mutation, and a correction template was used for homology-directed repair (HDR) ( Figure 3 A). IPSCs were nucleofected with two plasmids expressing the D10A Cas9 nickase and paired guide RNAs or a single plasmid expressing wild-type Cas9 and a single guide RNA. Cells were grown in medium containing G418 for 2 weeks post nucleofection. Individual colonies were picked, expanded, and genotyped by PCR ( Figure 3 B, top). The efficiency of CRISPR/Cas9-mediated JAK3 gene correction is shown in Figure 3 C. Three clones from wild-type Cas9 + gRNA #1, three clones from wild-type Cas9 + gRNA #2, and six clones from D10A Cas9 nickase + paired gRNAs #1 and #2 were further verified by Sanger sequencing. In 12 sequenced clones, two homozygous corrected clones (one clone from D10A Cas9 nickase + paired gRNA #1 and #2 and one clone from wild-type Cas9 + gRNA #1) and ten heterozygous corrected clones were identified ( Figure 3 D). Restoration of JAK3 gene expression was demonstrated by RT-PCR (JAK3 mRNA) ( Figure 3 B, lower-left panel) and western blot (JAK3 protein) ( Figure 3 B, lower right).

Forced expression of Bcl2 rescues T but not B or NK cell development in γc-deficient mice (). Transplantation of Jak3 KO mice with Bcl2-expressing Jak3 KO bone marrow cells also improves peripheral T cell numbers (). To determine whether overexpression of BCL2 rescues the T cell developmental defects of human JAK3-deficient cells, we transduced in-vitro-derived, JAK3-deficient CD34cells with a lentivirus containing a BCL2-2A-GFP polycistron driven by the EF1a promoter. After transduction, CD34cells were plated onto OP9-DL4 monolayers and assayed for NK and T cell markers at T cell induction day 28. No CD3CD16CD56NK cells were found in GFP(JAK3; BCL2 low) or GFPcells (JAK3; BCL2) ( Figure 2 C). These findings are consistent with reports demonstrating that the absence of NK cells in γc-deficient and Jak3-deficient mice is due to the lack of functional IL-15 signaling () and is independent of Bcl2-mediated anti-apoptosis. CD3cells were only detected in GFP(JAK3; BCL2) cells suggesting that BCL2 released the developmental block at the DN stage in JAK3-deficient cells. Interestingly, a second developmental arrest was evident at the DP stage; no further differentiation of CD8CD4DP cells was observed in GFPcells ( Figure 2 C). These data are consistent with recent demonstrations that signaling by intrathymic IL-7 is necessary for CD8 lineage specification of DP thymocytes ().

The profound defects in lymphocyte development of JAK3-deficient cells can be explained by the absence of IL-7 signaling, which plays an important role in lymphoid progenitor survival () and differentiation (). IL-7/JAK3 signaling maintains thymocyte homeostasis by regulating the BCL2 family of apoptotic regulators. Thymocytes and peripheral T cells from Jak3 KO mice have a high apoptotic index in part through selectively elevating Bax, a pro-apoptotic factor, and by reducing expression of Bcl2, an anti-apoptotic factor (). Similarly, we observed an increase in apoptosis of in-vitro-derived human JAK3-deficient cells compared to controls at T cell induction day 10 (9%–2.2%) and T cell induction day 17 (7%–1.9%) ( Figure 2 A). Consistent with this phenotype, BAX levels were increased and BCL2 levels were reduced in JAK3-deficient cells compared to controls ( Figure 2 B).

The transitions of early T cell progenitors (ETPs) from CD4CD8double-negative (DN) → CD4CD8double-positive (DP) → CD4single-positive (SP) or CD8single-positive (SP) T cells are directed by precise activation and repression of specific transcription factors () ( Figure 1 B). In control cells ( Figure 1 C), silencing of PU.1 gene expression and induction of GATA3 and BCL11B gene expression direct early hematopoietic progenitors to proceed to the onset of T lineage commitment. In JAK3-deficient cells, expression of the PU.1 gene was not completely silenced, induction of the GATA3 and BCL11B genes was significantly lower than controls, and T cell specification was severely limited. However, low-level expression of the T cell-specific genes RAG1, RAG2, and PTCRA suggests that JAK3-deficient cells can progress at low efficiency to an early T cell progenitor stage. Jak3 knockout (KO) mice have a small thymus due to a block in thymocyte differentiation at the CD4CD8double-negative 2 (DN2) stage prior to productive TCR rearrangement. Interestingly, some residual thymocytes in Jak3 knockout (KO) mice develop into mature T cells and reconstitute the peripheral population (); this does not occur in human JAK3-deficient patients. To further understand the developmental defects resulting from JAK3 deficiency in humans, we assayed T cell lineage commitment and maturation in JAK3-deficient cells compared to normal JAK3 WT controls. IPSC-derived CD34cells were plated onto OP9-DL4 monolayers, and cells were harvested and analyzed for lymphocyte markers at T cell induction day 28. No CD4CD8DP, CD4SP, or CD8SP T cells were detected in JAK3-deficient cells. Moreover, CD3 and TCR αβ were not significantly detected in JAK3-deficient cells ( Figure 1 D). The complete absence of CD4CD8cells indicates that human JAK3-deficient cells arrest at the CD4CD8DN stage. Low expression of the DN3-associated genes PTCRA, RAG1, and RAG2 suggests that human JAK3-deficient cells arrest at or before the DN2 stage. Unlike the results in Jak3 KO mice in which residual thymocytes develop past the CD4CD8DN stage into mature T cells that partially reconstitute the peripheral blood system, our data demonstrate a complete block of T cell development in human JAK3-deficient cells. This block is consistent with the absence of mature T cells in peripheral blood of JAK3-deficient human SCID patients.

IPSCs were generated from skin keratinocytes () of a SCID patient homozygous for a C > T nucleotide substitution in exon 14 of the JAK3 gene. This mutation replaces a CGA codon (arginine at 613) with a TGA stop codon (p.R613X). The 4-month-old patient presented with a TNKclinical phenotype (see Experimental Procedures ). To determine whether this SCID phenotype can be recapitulated in vitro, we attempted to differentiate patient-specific iPSCs to T lymphocytes using our previously published two-step OP9 and OP9-DL4 system (). JAK3-deficient iPSCs grew at a rate comparable to control iPSCs derived from healthy donors, and these iPSCs efficiently differentiated into CD34hematopoietic progenitors (HPs) on OP9 stromal cell monolayers. However, when the JAK3-deficient, iPSC-derived CD34HPs were plated onto OP9-DL4 stromal monolayers, NK and T cell differentiation was dramatically decreased compared to controls ( Figure 1 A). Only a small population of CD7CD16CD56T cells or CD7CD16CD56NK cells was observed at T cell induction day 14 ( Figure 1 A).

Discussion

Much has been learned about lymphopoiesis in the past 2 decades from naturally occurring immunodeficient mice and from knockout and knockin mouse models. These data have provided fundamental knowledge about the mechanisms involved in lymphocyte development and activity. In humans, the phenotype of lymphocytes in the peripheral blood of SCID patients has been well described, but studies on critical steps of lymphoid commitment and thymocyte development have been difficult to perform. Access to bone marrow and thymocyte samples from untreated patients with SCID is challenging since these conditions are rare and infants typically present with life-threatening infections requiring urgent HSC transplantation to survive. The strategy that we describe for studying human SCID bypasses these restrictions; large numbers of hematopoietic progenitors can be produced from patient-specific iPSCs in vitro, and the mechanisms responsible for immunodeficiency can be precisely determined. In this study, we demonstrate that T cell development in human JAK3-deficient SCID is completely blocked before or at the CD4–CD8– (DN2) stage. Interestingly, forced expression of BCL2 enhances survival of DN cells, which further differentiate into DP thymocytes. However, DP thymocytes fail to mature to SP T cells, and this defect may result from the absence of IL-7/JAK3 signaling.

We also demonstrate that correction of the human JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores the differentiation potential of early T cell progenitors. Corrected progenitors are capable of producing NK cells and mature T cell populations expressing a broad TCR repertoire. Whole-genome sequencing of one homozygous and two heterozygous corrected iPSC lines demonstrates that no mutations (SNVs nor indels) are introduced into 1,450 potential off-target sites, suggesting a strong specificity for CRISPR/Cas9-directed gene correction. In summary, these studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies and other monogenic disorders. For gene therapy, we envision transplantation of the CD34+ cells that are generated in the first phase of in vitro culture. These cells include early multipotent hematopoietic progenitors that generate all myeloid and erythroid cells in colony forming assays in addition to the lymphoid cells that we describe here. Our results suggest that there are no intrinsic defects in lineage specification of early hematopoietic progenitors produced in vitro. However, we have not been able to generate all of these lineages after transplantation into immunodeficient (NSG) mice. These results suggest that human hematopoietic progenitors produced in vitro do not home to mouse bone marrow niches that support self-renewal. However, these early progenitors may be incorporated into human bone marrow niches to which endogenous progenitors naturally home. After safety studies are completed in NSG mice, phase 1 clinical trials will be required to determine whether these early progenitors are capable of engraftment and sustained reconstitution of multilineage hematopoiesis in human patients.