Loss of Zbtb32 in NOD mice does not significantly alter T cell responses.

Mice

Non-Obese Diabetic mice (NOD, JAX #001976) were initially obtained from The Jackson Laboratory, Bar Harbor ME, and bred in the facility at NIH. Additional NOD females were regularly delivered every two weeks to compensate for the loss of breeders due to diabetes in the wildtype NOD colony. Approximately 630 NOD.Zbtb32^-/- mice (about 90 litters with an average liter size of 7) were bred during the characterization the mutant strain. All mice were housed in a specific pathogen free vivarium with a 12 h on/12 h off light cycle (6 am on and 6 pm off). The care, use, and disposition of all mice used in this study were reviewed and approved (protocol K024-DEOB-16) by the Institutional Animal Care and Use Committee of the NIDDK, NIH. Diabetes was monitored and mice were considered diabetic on the first of two blood glucose level measurements above 250 mg/dl.

Generation of knockout

The NOD.Zbtb32^-/- genetic alteration was carried out as follows: NOD female mice (6–8 weeks old) were super ovulated and mated overnight with NOD male mice (>8 weeks old). Zygotes were harvested from the ampullae of super ovulated females and were placed in KSOM medium (Millipore, Billerica MA) before microinjection. Microinjection was performed in M2 medium (Sigma, St Louis, MO) using a micromanipulator (Narishige) and microscope (Nikon). The second exon of the Zbtb32 gene was targeted using the two following RNA guide sequences; 5'-UACAG UUAGC GGCUA GACUC-3' and 5'-CAAUC AUGGA UCCCC CAUUG-3'. Binding sites for single guide RNA (sg RNA) are indicated in Figure 1A. The modified Cas9n enzyme with nickase activity (System Biosciences) and sgRNA were co-injected into the pronucleus and cytoplasm of 238 zygotes (1^st round 37, 2^nd round 47, and 3^rd round 154) and resulted in a total of 35 pups. The final injection concentration in the mixture was 10 ng/μl Cas9n and 5 ng/μl of each sgRNA. After injection and incubation in 5.5% CO₂ at 37°C overnight for 24 h, surviving 2-cell stage embryos were transferred to female NOD pseudopregnant recipients via oviduct transfer.

Figure 1. Confirmation of CRISPR-mediated knockout of Zbtb32.

The CRISPR/Cas9 technique was used to target exon 2 of the Zbtb32 gene directly in NOD mice. A portion of the Zbtb32 gene was deleted and a frameshift introduced in NOD mice using the CRISPR/Cas9 technique, as illustrated here (A). The scissors represent the location of cutting based on sgRNA binding and black arrows indicate binding sites for the standard primers, which were used for both sequencing and genotyping. The location of the deletion maps onto the beginning of exon2 of the wildtype mRNA (B). Non-coding portions are shown as thinner than the coding portions. The CRISPR/Cas9-treated embryos develop into mice with genetic mosaicism of the Zbtb32 gene in the F0 generation. Four genetically heterogeneous females (indicated with black arrows) were examined further before one mutant allele was chosen to establish the NOD.Zbtb32^-/- colony (C). The targeted deletion of Zbtb32 removed 146 bp from exon 2 as shown by genetic screening of knockout, heterozygous, and wildtype animals in the F2 generation (D). The loss of the Zbtb32 protein after CRISPR-mediated deletion was confirmed by Western blotting extracts from stimulated CD4⁺ splenocytes (E) (from three biologically independent replicates).

Characterization of CRISPR/CAS9n genetic alterations

Genomic DNA was extracted from tail snips of the 35 21 day-old F0 mice using the DNeasy blood and tissue kit (Qiagen). The target region was PCR amplified by PuReTaq Ready-To-Go PCR beads (GE Healthcare) followed by IndelCheck CRISPR/TALEN insertion or deletion detection system (GeneCopoeia). Successful genetic editing of the Zbtb32 gene was observed in 15 of the 35 pups. Genomic DNA from pups with evidence of deletion was further PCR amplified and subsequently cloned by TOPO T/A subcloning (Invitrogen, Carlsbad, CA) followed by DNA sequencing. Compound heterozygous null founders were bred with wildtype NOD animals to provide four potential alleles (named A, B, C, and D) for the creation of a NOD.Zbtb32^-/- colony. All four mutant alleles were sequenced to verify a successful deletion (Supplementary Table 1). As expected, the mutant alleles displayed a variety of deletion sizes due to differential DNA repair. Sibling crosses with matching mutant alleles were set up but, only mice carrying the mutant B allele were reliable breeders. Therefore, all NOD.Zbtb32^-/- mice described in this paper are homozygous for the B allele.

Genotyping and sequencing

For routine genotyping and sequencing, we used the following PCR primers: forward 5’-AGCTG GCCTT TGGCT TAGTT-3’ and reverse 5’-CAAAG GTGGA AGGGC TTATG-3’. Binding sites for these primers in relation to the CRISPR/Cas9 deletion are indicated in Figure 1A. PCR conditions followed a typical 3-temp program; 95°C for 5 min, [95°C 30s, 55°C 30s, 72°C 45s] repeat 34 times, and finally 72°C for 10 min. The PCR results were run out on a 2% agarose gel. Single pass DNA sequencing services (ACGT, Inc., Germantown, MD) were carried out on genomic DNA using the reverse primer.

Western blotting

During dissections, mice were euthanized by CO2 asphyxiation and then the spleen was removed and placed in ice-cold PBS. Splenocytes were negatively enriched for CD4⁺ T cells using magnetic beads via the mouse CD4⁺ T Cell Isolation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany) and then were incubated with Armenian Hamster anti-mouse CD3 (clone 145-2C11, Biolegend cat#100301) and Syrian Hamster anti-mouse CD28 (clone 37.51, Biolegend cat#102111) for either 14 h or 24 h at 37°C in T-cell Media (RPMI-1640+10% FBS+L-glutamine +Penn/Strep). The cells were then pelleted and lysed used RIPA buffer supplemented with Protease Inhibitor Cocktail (Roche, Indianapolis, IN). A total of 35 µg of protein was loaded into a NuPAGE 4–12% Bis-Tris gel, transferred onto nitrocellulose, blocked in 5% milk for 1 hour at 25°C, and blotted for Zbtb32 using rabbit anti-mouse sc-25358 (Santa Cruz Biotech, Santa Cruz CA) at a 1:500 dilution overnight at 4°C. The membrane was then washed prior to the addition of goat-anti-rabbit HRP (Jackson Immunoresearch cat#111-035-003) at a dilution of 1:5,000 dilution at 1 hour at 25°C. The membrane was then stripped, reblocked and reblotted for Lamin B using goat anti-mouse sc-6217 at a 1:5,000 dilution for 1 hour at 25°C. The membrane was then washed prior to the addition of donkey anti-goat HRP (Jackson Immunoresearch cat#705-035-003) at a dilution of 1:5,000 dilution for 45 minutes at 25°C. All protein bands were imaged on a BioRad ChemiDoc CCD system and processed with the BioRad Image Lab 6.0 software.

Ex vivo stimulations and cytokine staining

Splenocytes and lymphocytes were stimulated with anti-mouse CD3 (145-2C11; BioLegend, San Diego, CA). A total of 5×10⁵ cells were incubated for 18 hours at 37°C in T-cell media with either 0, 10, 100, or 1000 ng of anti-CD3 in a 300 µl volume. For cytokine staining, cells were stimulated with PMA and Ionomycin in T-cell Media for 5 hours at 37°C. Brefeldin A was added to all cells for the final 4 hours of stimulation. Cells were then fixed and stained for flow cytometry. FMO staining controls were performed using a mixture of both unstimulated and stimulated cells.

Flow cytometry

All cells were washed in Wash Buffer [PBS+2% FBS] and then blocked with LEAF purified anti-CD16/32 (clone 93) on ice for 30 minutes prior to staining for flow cytometry. The Foxp3 (FJK-16s) antibodies were purchased from eBioscience (San Diego, CA). Foxp3 antibodies were diluted at 1:50 in Wash Buffer for cell staining, while all other antibodies were diluted at 1:100 in Wash Buffer. Staining was carried out for a minimum of 1 hour at 4°C. Antibodies purchased from Biolegend (San Diego, CA) are listed alongside their clone and Antibody Registry numbers. We used antibodies raised against mouse B220 (RA3-6B2, RRID:AB_11203907), CD1d (CD1.1, RRID:AB_2715919), CD4 (GK1.5, RRID:AB_312690), CD5 (53-7.3, RRID:AB_312736), CD11b (M1/70, RRID:AB_312798), CD8 (53-6.7, RRID:AB_2561352), CD19 (6D5, RRID:AB_130884), CD23 (B3B4, RRID:AB_10060129), CD25 (PC61, RRID:AB_2562611), CD43 (S11, RRID:AB_2563698), CD44 (IM7, RRID:AB_2621762), CD62L (MEL-14, RRID:AB_10555750), CD69 (H1.2F3, RRID:AB_10683447), CD335 (29A1.4, RRID:AB_10827686), IFNγ (XMG1.2, RRID:AB_469504). Cell viability was assessed by Fixable Live/Dead Aqua staining (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. Cell proliferation was measured using the Cell Trace Violet Kit (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. For cell fixation, permeabilization, and intracellular staining, we utilized the Intracellular Fixation & Permeabilization Buffer Set from eBiosciences. All samples were run on a BD Biosciences LSR-II cytometer and later analyzed using FlowJo v9.4.1. For analysis, CD44 expression was measured by mean fluorescence intensity (MFI) rather than by the percentage of positive expression.

Statistical analysis

Statistical analysis for all experiments was carried out using GraphPad Prism v5. Where appropriate, statistical significance was calculated using either a Kaplan-Meier survival method (for disease incidence), or a two-way ANOVA tests for independent samples with post hoc Bonferroni comparisons for each possible pair of groups (used for all other data). Any significant differences (P < 0.05) between the two control strains are denoted with an asterisk. All dot plots show exact data points with a horizontal line denoting the average. All histograms show the average and standard deviation for the data.

Using CRISPR techniques to generate knockout mutations directly in the NOD genetic background

The NOD mouse is an important model for examining spontaneous and chronic autoimmune disease, but it has been difficult to obtain genetically modified NOD mice due to the lack of a robust NOD ES cell line¹⁹. Directly targeting mutations into the NOD genetic background via CRISPR gene editing in NOD embryos is now possible²⁰. Matching sgRNAs in exon 2 of Zbtb32 (Figure 1A and B) and CAS9n were injected into 2 cell-stage embryos. Using the CAS9n with nickase activity ensures that an insertion or deletion can only occur if CAS9n binds to both sgRNAs, vastly reducing off-target events²¹. Four female mice carrying a suitable deletion were selected to be potential F0 founders (indicated in Figure 1C) and were crossed with wildtype NOD mice to produce heterozygous mice in the F1 generation. Two of these potential F0 founders were found to be germline compound heterozygous for the loss of Zbtb32, providing up to four potential alleles (listed in Supplementary Table 1 as alleles A, B, C, and D) for the creation of a NOD.Zbtb32^-/- colony. Sibling crosses with matching mutant alleles were set up using the F1 generation. Because mice carrying the mutant B allele were the most reliable breeders, all NOD.Zbtb32^-/- mice described in this paper are homozygous for the B allele containing a 146 bp frameshift deletion.

We designed PCR primers to serve for both routine genotyping purposes (demonstrated in Figure 1D) and as sequencing primers. Genomic sequencing on select NOD.Zbtb32^-/- mice from multiple generations verified that the deletion within the Zbtb32 gene was stable (Supplementary Figure 1). In wildtype NOD mice, Zbtb32 was not detectable at baseline in resting CD4+ lymphocytes, but could be detected by Western blot as early as 14 hours post anti-CD3/CD28 stimulation (Figure 1E). No Zbtb32 protein was observed in NOD.Zbtb32^-/- mice at either baseline or post-stimulation (Figure 1E).

Diabetes incidence in NOD.Zbtb32^-/- mice

To test the hypothesis that absence of Zbtb32 would increase diabetes pathogenesis in NOD mice, we monitored diabetes via blood glucose levels in NOD.Zbtb32^-/- mice and their control littermates for up to 35 weeks for both female (Figure 2A) and male mice (Figure 2B). NOD mice normally spontaneously develop hyperglycemia, between 12 and 30 weeks of age due to autoimmune destruction of the insulin secreting β-islet cells within the pancreas. The diabetes incidence rates showed no statistical differences between NOD.Zbtb32^-/- mice and their wildtype littermate controls when using a standard Kaplan-Meir survival test, but the male NOD.Zbtb32^-/- trended toward a higher than expected total diabetes incidence (Figure 2C). Therefore, the trend in higher male disease incidence may indicate a mild phenotype in NOD.Zbtb32^-/- mice may impact disease onset over long timescales.

Figure 2. Diabetes incidence in NOD.Zbtb32^-/- mice.

The onset of Type 1 diabetes was monitored in our NOD.Zbtb32^-/- colony by weekly blood glucose monitoring for both female (A) and male (B) mice. Disease incidence rates were followed for littermate wildtype, and homozygous knockout mice. Disease incidence rates for males and females were plotted in (C).

Ex vivo T cell numbers and activation levels are not significantly altered in NOD.Zbtb32^-/- mice

Potential differences between wildtype and Zbtb32^-/- lymphocyte phenotype of both male and female mice at 8 weeks of age were tested using flow cytometry. Activation markers on Foxp3⁺ regulatory T cells (Tregs), conventional CD4⁺ T cells (Tcons), and CD8⁺ T cells were measured without exogenous stimulation. As expected, higher activation was measured in the pancreatic-draining lymph nodes (pLNs) (Figure 3) compared to the spleen (Supplementary Figure 2), consistent with an autoimmune reaction to antigens acquired from pancreatic tissue. However, no statistically significant differences were observed between the NOD.Zbtb32^-/- mice and their control littermates for percent CD25⁺ (Figure 3A), CD69⁺ (Figure 3B), or CD44 MFI (Figure 3C) (and Supplementary Figure 2A – C). T cells from the NOD.Zbtb32^-/- pLN, but not the spleen were more variable than the wildtype for these phenotypes, and in some comparisons trend higher (e.g. elevated CD25 expression in female CTLs seen in Figure 3A), suggesting a mild activation phenotype.

Figure 3. Activation markers on lymphocytes from pancreatic draining lymph node of Zbtb32^-/- mice.

Lymphocytes from the pancreatic draining lymph node of male and female NOD and NOD.Zbtb32^-/- mice were stained for activation markers. Flow cytometry was used to determine cell surface expression of CD25 (A) CD69 (B) and CD44 (C) on CD4⁺ Foxp3⁺ (Tregs), CD4⁺ Foxp3^- (Tcon) and CD8⁺ T cells. The data were combined from two independent repeats of this experiment.

In vitro stimulation of splenocytes is not significantly altered in NOD.Zbtb32^-/- mice

In previous reports describing Zbtb32 gene deletion in other genetic backgrounds, reported phenotypes included T-cell hypersensitivity, increased proliferation, and increased cytokine production^10–12,14. We therefore carried out parallel experiments using splenocytes from the NOD.Zbtb32^-/- mice. Splenocytes from both NOD.Zbtb32^-/- mice and NOD wildtype mice were stimulated with anti-CD3, and cell activation was assessed after 18 h by staining for surface CD69 (Figure 4A–C), CD44 (Figure 4D–F) and CD25 (Supplementary Figure 3A–C). T cell activation markers increased with anti-CD3 stimulation, but no significant differences between wildtype and Zbtb32^-/- lymphocytes were observed. Levels of cell proliferation after 48 hours of anti-CD3 stimulation, as measured by dilution of Cell Trace Violet dye, were also the same between NOD wildtype and Zbtb32^-/-(Supplementary Figure 3D and E). We next measured cytokine production by stimulating splenocytes with PMA and Ionomycin, and detecting IFNγ production in CD4⁺, CD8⁺, and Nkp46⁺ cells via intracellular cytokine staining. In all cases, the stimulation with PMA and Ionomycin produced the expected production of IFNγ, with higher expression in CD8⁺ and Nkp46⁺ cells. However, we did not find statistically significant differences between NOD.Zbtb32^-/- splenocytes and their matching controls (Figure 4G–I). Therefore, unlike other Zbtb32^-/- strains, ex vivo T cell responses from NOD.Zbtb32^-/- mice were not significantly altered.

Figure 4. Activation markers and cytokine production in Zbtb32^-/- splenocytes after ex vivo stimulation.

Freshly isolated splenocytes from female NOD.Zbtb32^-/- mice and control littermates were stimulated with the indicated dosages of anti-CD3 for 18 h. Surface expression of CD69 (A–C) and CD44 (D–F) was measured on CD4⁺, CD8⁺, and NKp46⁺ splenocytes. For IFNγ staining, splenocytes from female NOD.Zbtb32^-/- mice and their control littermates were stimulated with PMA and Ionomycin for 4 hours (G–I). The percentage of cells positive for IFNγ in CD4⁺ (G), CD8⁺ (H), and NKp46⁺ cells (I) is shown. The data were combined from two independent repeats of this experiment.

No significant changes in peritoneal cavity B cells in NOD.Zbtb32^-/- mice

Because the T cell and diabetes phenotypes did not match the expected result based on overexpression of Zbtb32 in T cells⁶, potential effects of Zbtb32 deletion on other cell types was considered. B1 cells, which primarily reside in the peritoneal cavity, display a constitutively high expression of Zbtb32^22,23. A related transcription factor, Zbtb20, was recently implicated as a regulator of terminal differentiation in germinal center B cells²⁴. The authors proposed that either Zbtb20 or Zbtb32 may play a critical role in either B1 cell proliferation or differentiation. B1 cells can be further divided into B1a and B1b cells based on their surface expression of CD5²⁵. In NOD mice, B1 cells can be identified as CD19⁺B220⁺CD11b⁺CD43⁺, with CD5⁺ and CD5^- subpopulations being labeled as B1a and B1b, respectively (Figure 5). Since prior reports had indicated that Zbtb32 can regulate early B cell proliferation²⁶, B1 populations in the peritoneal cavity of NOD.Zbtb32^-/- mice and their wildtype controls were examined at 4 weeks of age (Figure 5). NOD.Zbtb32^-/- mice trended toward fewer total B cells in their peritoneal cavity. But no statistically significant differences were observed in the proportion of B1 cells among all CD19⁺ cells between wildtype NOD and NOD.Zbtb32^-/- mice (analysis not shown). Therefore, the mild diabetes phenotype and the absence of the expected T cell activation phenotype are unlikely to be due to effects on B1 cells.

Figure 5. B cell populations within the peritoneal cavity.

The number of B cells in the peritoneal cavity of 4 week-old mice were measured, including all CD19⁺ cells, all B1 cells, and the subpopulations of B1a and B1b cells. The data were combined from two independent experiments.