Zebrafish Model of Severe Combined Immunodeficiency (SCID) Due to JAK3 Mutation

JAK3 is principally activated by members of the interleukin-2 receptor family and plays an essential role in lymphoid development, with inactivating JAK3 mutations causing autosomal-recessive severe combined immunodeficiency (SCID). This study aimed to generate an equivalent zebrafish model of SCID and to characterize the model across the life-course. Genome editing of zebrafish jak3 created mutants similar to those observed in human SCID. Homozygous jak3 mutants showed reduced embryonic T lymphopoiesis that continued through the larval stage and into adulthood, with B cell maturation and adult NK cells also reduced and neutrophils impacted. Mutant fish were susceptible to lymphoid leukemia. This model has many of the hallmarks of human SCID resulting from inactivating JAK3 mutations and will be useful for a variety of pre-clinical applications.


Introduction
Cytokines and their downstream signaling components play major roles in regulating blood and immune cell development and function [1,2]. Janus kinases (JAKs) are receptor-associated tyrosine kinases that are pivotal in cytokine receptor-mediated signal transduction, such that cytokine binding triggers their activation thereby facilitating stimulation of downstream pathways controlling critical cell processes such as proliferation, survival and differentiation [3]. Disruption of JAKs has been associated with numerous blood and immune diseases [4].
Zebrafish have become a pivotal platform for the study of blood and immune cells [16], and share high conservation of cytokine receptor signaling components, such as equivalent IL-2Rγc [17] and JAK3 [3] proteins. It therefore represents a highly suitable organism in which to model relevant human diseases, including immunodeficiency [18][19][20] and leukemia [21,22]. This study describes the generation of zebrafish harboring jak3 mutations similar to those seen in human SCID and the characterization of the blood and immune cells within these mutants throughout the life-course.

Zebrafish Husbandry
Zebrafish were maintained using standard husbandry practices [23], following National guidelines of animal use and care. Embryos were obtained by manual spawning of adult fish and were subsequently maintained at 28.5 • C in a Petri dish containing aquarium water supplemented with 0.003% 1-phenyl-2-thio-urea (PTU) at 8 h post fertilization (hpf) to inhibit pigmentation. All studies involving animals were performed under approval from the Deakin University Animal Welfare Committee.

Whole-Mount In Situ Hybridization (WISH)
Embryos were collected at specific time points, anesthetized with 0.4 mg/mL benzocaine and fixed in 4% (w/v) paraformaldehyde (PFA) in phosphate-buffered saline (PBS) before storing them at 4 • C. Embryos were subsequently hybridized using digoxygenin (DIG)-labelled RNA probes as described previously [25]. Images were taken using an Olympus MVX10 monozoom microscope with a 1× MVXPlan Apochromat lens (NA = 0.25) and DP74 camera and quantitation performed by manual counting of dispersed individual cells or by measuring the area of the thymus utilizing CellSens Dimensions 1.6 software (Olympus, Notting Hill, Australia). Data from approximately 30 embryos were collected and analyzed for significance using a Student t-test with data tested for normality and Welch's correction performed where necessary.

Ex Vivo Analyses
Cytospin preparations of adult blood were stained with Giemsa (Sigma, St. Louis, MO, USA), imaged using a Leica DM E microscope with a 100× oil objective (NA = 1.25) and an Olympus SC50 camera and differential counts performed. Zebrafish that developed lymphocytic leukemia were fixed in formalin, followed by paraffin embedding, sectioning and staining with Hematoxylin and Eosin and imaged using an Olympus BX46 clinical microscope with a UPLFLN (PH) Plan Semi Apochromat (OFN 26.5) and DP22 camera, utilizing Olympus CellSens Entry 3.2 (Build 23706) software.

Survival Analysis
Adult fish were monitored by regular visual inspection with relative survival displayed as a Kaplan-Meier curve and statistical significance determined using a log-rank (Mantel-Cox) test.

Generation of SCID-Related Jak3 Mutant Zebrafish
A variety of inactivating mutations of human JAK3 have been identified in autosomal recessive SCID patients, with the pseudokinase domain (PKD) being a hot spot for such mutations [13,27]. To generate similar mutations in zebrafish Jak3, CRISPR/Cas9-mediated genome editing was employed to target exon 13 of the zebrafish jak3 gene that encodes the PKD ( Figure 1B). One-cell stage zebrafish embryos were injected with Cas9 mRNA and in vitro transcribed gRNA specific for this exon ( Figure 1C). These were raised to adulthood and their progeny screened by HRM analysis followed by sequencing. This identified two mutant alleles, mdu9 and mdu10, which carried an 11 bp insertion and a 4 bp deletion at the target site, respectively. Both of these mutations introduced a premature stop codon leading to a Jak3 protein truncated in the PKD at the same amino acid ( Figure 1C). Another allele, mdu11, carried a 2 bp deletion, leading to a frameshift at the same site. Founder fish carrying these alleles were out-crossed twice to dilute any potential off-targeting, with F2 heterozygous mutants in-crossed to generate F3 wild-type, heterozygous and homozygous jak3 mutants for further analysis. . Nucleotide sequence of zebrafish homozygous for wild-type (wt) and mutant (mdu9 and mdu10) alleles of jak3, with their protein translations displayed below in black text and the CRISPR/Cas9 target site shown above. The mdu9 allele represents an 11 bp insertion (orange box) and the mdu10 allele a 4 bp deletion (purple box), both resulting in frameshifts that introduce a stop codon at the equivalent location within the pseudokinase domain.

SCID-Related Jak3 Mutation Disrupts Embryonic Lymphopoiesis
The effect of Jak3 mutations on embryonic hematopoiesis was analyzed using WISH with specific gene markers. Homozygous jak3mdu9/mdu9 mutants showed significantly reduced expression of ikzf1, a marker of early T lymphocyte progenitors in the developing thymus [28], at 3.5 dpf compared to jak3 wt/wt siblings (Figure 2A-C). This decrease in thymus staining was sustained at 5 dpf ( Figure 2D-F), at which time reduced expression was also observed for rag1 ( Figure 2G-I) and tcra ( Figure 2J-L), markers of more mature T lymphocytes [29,30]. However, there was no significant difference in the number of cells expressing lyz ( Figure 2M-O), a leukocyte marker [31], or mpo ( Figure 2P-R), a neutrophil marker [32], between jak3wt/wt and jak3mdu9/mdu9 embryos. Expression of rag1 was similarly reduced in homozygous jak3mdu10/mdu10 mutants, with heterozygous jak3wt/mdu10 embryos similar to wild-type siblings ( Figure S2A-D), confirming the recessive nature of this mutation.

SCID-Related Jak3 Mutation Perturbs Larval Lymphopoiesis
The effect of Jak3 mutations on zebrafish larval lymphopoiesis was investigated at 28 dpf, when B cell development has commenced [33]. RT-PCR using primers specific for T cell and B cell rearrangements [34,35] showed normal T and B cell rearrangement in wild-type jak3 wt/wt individuals, whereas jak3 mdu9/mdu9 mutants showed no T cell rearrangement and variable B cell rearrangement ( Figure 3A). Homozygous jak3 mdu10/mdu10 larvae displayed a disruption in both T and B cell rearrangements ( Figure S2E). Figure 3. Effect of SCID-derived JAK3 mutations on zebrafish larval lymphopoiesis. Analysis of immune and blood cells in 28 dpf wild-type (wt/wt) and mutant (mdu9/mdu9) jak3 larvae using RT-PCR with primers specific for T cell receptor (TCR) β-chain (v(d)j-cβ vb1.5, vb12, vb14.5) and B cell Ig heavy chain (igvh1, igvh4) rearrangements along with actb as a control (n = 2) (A), noting that RT-negative controls yielded no products, or qRT 2 -PCR analysis with the indicated hematopoietic cell markers (B). Data is represented as relative fold-change compared to homozygous wild-type (wt/wt) larvae with mean and SEM shown in red and level of statistical significance of normalized Cq values (relative to control actb) indicated (** p < 0.01, * p < 0.05, ns not significant; n = 3).

SCID-Related Jak3 Mutation Perturbs Adult Lymphopoiesis
The impact of Jak3 mutations on adult zebrafish lymphopoiesis was first investigated by cytological analysis of peripheral blood. Differential counting revealed a significant reduction in circulating lymphocytes and an increase in neutrophils in jak3 mdu9/mdu9 compared to jak3 wt/wt fish ( Figure 4A-C). FACS analysis revealed of adult kidney cells demonstrated a significant decrease in lymphocytes, with myeloid cells also affected ( Figure 4D-I).
The adult kidney was further analyzed for expression of key cell lineage markers, which revealed significant reduction in the expression of HSC (lmo2), T cell (cd4, cd8, tcra, tcrb, rag1), NK cell (nccrp1, nkld) and neutrophil (mpo) markers in jak3 mdu9/mdu9 compared to jak3 wt/wt adults, with markers of early B cell (cd79a, pax5), macrophages (mpeg1.1) [38] and RBC (hbba1) not significantly altered ( Figure 4J). Interestingly, expression of mature B cell markers (ighm, ighd) was significantly reduced in jak3 mdu9/mdu9 adults. Despite this, Jak3 mutants were generally healthy, with similar survival to wild-type fish ( Figure 4K). (J) Analysis of hematopoietic cell lineages in adult kidney from wild-type and homozygous mutant fish using qRT 2 -PCR analysis with the indicated lineage cell markers. Data is represented as relative fold change compared to wild-type with mean and SEM shown in red and statistical significance of normalized Cq values (relative to control actb) indicated (*** p < 0.001, ** p < 0.01, * p < 0.05, ns not significant; n = 6). (K) Survival analysis of wild-type and mutant jak3 fish at each week post fertilization (wpf) displayed as a Kaplan-Meier plot (wt/wt, n = 70, mdu9/mdu9, n = 75).

SCID-Related Jak3 Mutants Are Susceptible to Lymphocytic Leukemia
From around 12 months of age Jak3 mutant fish developed visible signs of illness, including skin lesions and tumors affecting around 10% of individuals. Histological analysis revealed invasive, multicentric lymphoid neoplasms, with densely cellular and non-encapsulated neoplastic masses infiltrating the entirety of the fish, variably effacing multiple organs, including the brain ( Figure 5A,B), liver ( Figure 5C,D), kidney ( Figure 5E,F) and intestine ( Figure 5G,H). The monomorphic round neoplastic cells were arranged in sheets, supported by minimal fibrovascular stroma, and possessed basophilic cytoplasm with distinct borders and singular ovoid nuclei of stippled chromatin and more than one deeply eosinophilic nucleoli, characteristic of lymphocytic leukemia.

Discussion
Members of the IL-2R family represent the main cytokine receptors controlling lymphoid development and include the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 [39][40][41][42]. These all share the IL-2Rγc chain that signals via JAK3 and downstream effectors, such as STAT5, PI3K and IRS, to regulate the generation, proliferation and function of various immune cell populations [43]. Disruption of several of these signaling components has been shown to impair normal lymphoid development, with inactivating JAK3 mutations associated with autosomal-recessive SCID in humans and mice [13,14,44,45]. Zebrafish possesses many of the constituent chains of the IL-2R family, including two IL-2Rγc proteins [17] and a single Jak3 with high sequence conservation to its mammalian counterparts [3]. Zebrafish jak3 was found to be expressed in the thymus during embryogenesis ( Figure S1A-E), with higher expression observed in the adult spleen, kidney, and gills ( Figure S1E), consistent with the predominantly lymphoid expression of mammalian JAK3 [46][47][48]. Collectively, this suggested a similar biological role for JAK3 across vertebrates, which encouraged us to generate a zebrafish SCID model based on an appropriate jak3 mutant. SCID-causing JAK3 mutations are found throughout the JAK3 protein, [13,27] such as a non-sense mutations in the PKD [15] and frame-shift mutations in the SH2 [15], FERM [49] or kinase [50] domains. Mis-sense mutations have also been identified, including an E481G substitution mutation in the SH2 domain leading to a milder SCID phenotype [50] and a compound PKD C759R and SH2 domain non-sense mutation [50]. In this study the PKD of zebrafish Jak3 was targeted, with two independent mutant alleles identified each truncating at the same amino acid within this domain.
Human SCID caused by JAK3 mutation is characterized by severely reduced T and NK cells, but with B cells present, although with compromised functionality [14,15]. The zebrafish jak3 mutants displayed a severe reduction in T lymphocytes during embryonic hematopoiesis that continued into the larval stage. NK cells were reduced in the adult, with evidence of abrogated B cell maturation and some disruption of the neutrophil compartment. This is consistent with a study showing jak3 mutants have reduced early T cells but where the effects on B cells and NK cells or into adulthood were not characterized [20], and another solely reporting depletion of T and NK cells in the adult kidney [19]. The data presented here for the first time characterized the complete impact of zebrafish jak3-deficiency throughout the life-course.
The phenotype of human SCID due to JAK3 mutations is very similar to that observed in X-linked SCID (X-SCID) resulting from mutations in the IL2Rγc-chain [51]. Both Jak3 and Il-2rγc knockout mice also developed a form of SCID, although in this case with markedly reduced T, NK and B cells [44,45,[52][53][54]. Inactivating mutations in zebrafish Il2rγc.a resulted in a SCID phenotype, with reduced T and NK cells but normal B cells [18,55], like in humans. The variation in B cell numbers between mouse and human mutants has been attributed to IL-7 being a pre-B cell growth factor in mice but not in humans [56]. Patients harboring mutations in IL-7 receptor alpha chain have a T-B+NK+ SCID phenotype [56,57]. Mice with IL-7 and IL-7R deficiency possessed reduced levels of T and B cells [58,59], with IL-7R deficient mice having normal NK cell levels [60]. Zebrafish carrying an inactivating mutation of Il-7 displayed reduced T cells, but unaffected B cells again similar to humans [20], with NK cells not characterized in that study. The difference between zebrafish Jak3 and Il2rγc.a mutants might be due to the duplicated Il2rγc.b receptor in zebrafish [17], which may play a role in B cell development and maturation.
The functional role of JAK3 in lymphopoiesis is well characterized. However, its role in myeloid cell development and differentiation is less explored. One publication has identified impaired myelopoiesis in Jak3-deficient mice, with the maturity of both neutrophils and monocytes impacted [61]. Another identified JAK3 as a primary response gene induced by granulocyte colony-stimulating factor (G-CSF) with overexpression of JAK3 associated with cell growth inhibition and terminal granulocytic differentiation [62]. This study has revealed disruption of neutrophil homeostasis in zebrafish jak3 mutants, being decreased in the kidney and increased in peripheral blood. Delineating the mechanistic details remains a worthwhile undertaking.
Notably, the zebrafish jak3 mutants survived to adulthood, which contrasts with the poor prognosis in human JAK3-SCID, with patients succumbing to the disease during early childhood if left untreated [63]. This probably reflects the greater dependence on innate compared to adaptive immunity in teleost fish, with adaptive immunity not established until 4 weeks of age [64], with several higher level aspects not present [65]. However, adult zebrafish jak3 mutants developed a malignant lymphoid leukemia, which invaded multiple organs like brain, kidney, liver and intestine. We were unable to definitively characterize this due to a lack of key reagents, but this observation is consistent with primary immune deficiency patients developing cancer, particularly hematological rather than solid tumors [66]. This suggests that the immunodeficiency caused by JAK3 mutation significantly impacts tumor immunity.

Conclusions
This study describes the generation and characterization of a zebrafish model of SCID due to Jak3 deficiency. These zebrafish mutants showed depletion of embryonic, larval and adult T cells, reduced B cell maturation and NK cell numbers in adults, which also displayed disrupted neutrophil homeostasis and susceptibility to lymphoid malignancy. It is anticipated that this line will enable further exploration of the role of Jak3 in the myeloid lineage, as well as enable studies examining the microbiota and facilitate xenotransplantation studies, as recently described for an IL-2Rγc model of SCID [18].