Fig 1.
Characterization of spontaneous CD4-deficient phenotype.
(a–b) Flow cytometry analysis of peripheral blood mononuclear cells from wild type (top) and β2m –/–mice with CD4-deficient phenotype (bottom). Peripheral blood cells were stained with anti-CD4, anti-CD8, H2-Kb, CD19, TCRβ, and I-A/I-E antibodies. Representative flow cytometry plots from 6 independent litters of mice that were analyzed are shown. n = 6–21 mice per group. (c) Flow cytometry analysis of thymocytes from β2m –/–mice (left) and β2m –/–mice with CD4-deficient phenotype (right). Thymocytes were stained with anti-CD4 and anti-CD8 antibodies. Representative flow cytometry plots from at least 4 independent experiments are shown. n = 6–44 mice per group. (d) Flow cytometry analysis of peripheral blood mononuclear cells from F1 mice (top) and F2 mice (bottom) from the breeding of CD4-deficient phenotype with wild type C57BL/6 mice. Peripheral blood cells were stained with anti-CD4, anti-CD8, H2-Kb, CD19, TCRβ, and I-A/I-E antibodies. Representative flow cytometry plots 2 independent experiments are shown. n = 4–8 mice per group.
Fig 2.
Identification of the mutation responsible for the CD4-deficient phenotype.
(a) Indication of the indel mutation leading to a frameshift in the CD4 mutant mice, resulting in a premature stop codon in the exon 7, identified through exome sequencing. Sequences of the exon 7 from both wild-type mice (top) and CD4 mutant mice (bottom) are shown, with the frameshift resulting in a stop codon highlighted in red. (b) Sanger sequencing of the CD4 gene from tail genomic DNA in wild-type mice (left) and CD4 mutant mice (right), confirming the presence of the mutation at this site. Representative sequencing data from 6 independent litters of mice that were analyzed are shown. n = 10–24 mice per group. (c) Flow cytometry analysis of 58 –/–cells transduced with wild type (top) or mutant CD4 gene (bottom) with a retroviral vector expressing BFP. 58 –/–cells were stained with anti-CD4 antibody and the expression of both BFP and CD4 was observed. Representative flow cytometry plots from 2 independent experiments are shown. (d) Flow cytometry analysis of BFP+ 58 –/–cells transduced with the CD4 mutant construct and untreated (top) or receiving the Cas9/sgRNA/repair nucleotide complex (bottom). 58 –/–cells were stained with anti-CD4 antibody and the expression of both BFP and CD4 was observed. Representative flow cytometry plots from 2 independent experiments are shown. (e–f) Flow cytometry analysis of inguinal lymph nodes from wild type (top) or CD4fs (bottom) mice after adoptive transfer of 3–4 million eFluor450 labeled cells from myelin-specific TCR transgenic mice. Mice were immunized with 100 μg MOG 35–55 in 5 mg/ml of CFA 24 h after cell transfer and lymph nodes collected 72 h after immunization. (e) Representative flow cytometry plots show the transferred Vα3.2+ Vβ11+ T cell population (left) and the dilution of eFluor450 as a measure of the proliferation of the transferred cells (right). (f) The proportion of Vα3.2+ Vβ11+ T cells in wild type (WT, n = 5) and CD4fs (n = 6) mice. **P = 0.0043 (Mann Whitney’s U test). Each circle represents one mouse. Data represented as median with 95% CI. The experiments were performed twice with similar results.
Table 1.
Quantification of T cell numbers and frequencies from wild type and CD4fs mice after adoptive transfer and immunization.