Prss16 is not required for T-cell development

Abstract

PRSS16 is a serine protease expressed exclusively in cortical thymic epithelial cells (cTEC) of the thymus, suggesting that it plays a role in the processing of peptide antigens during the positive selection of T cells. Moreover, the human PRSS16 gene is encoded in a region near the class I major histocompatibility complex (MHC) that has been linked to type 1 diabetes mellitus susceptibility. The mouse orthologue Prss16 is conserved in genetic structure, sequence, and pattern of expression. To study the role of Prss16 in thymic development, we generated a deletion mutant of Prss16 and characterized T-lymphocyte populations and MHC class II expression on cortical thymic epithelial cells. Prss16-deficient mice develop normally, are fertile, and show normal thymic morphology, cellularity, and anatomy. The total numbers and frequencies of thymocytes and splenic T-cell populations did not differ from those of wild-type controls. Surface expression of MHC class II on cTEC was also similar in homozygous mutant and wild-type animals, and invariant chain degradation was not impaired by deletion of Prss16. These findings suggest that Prss16 is not required for quantitatively normal T-cell development.

FIG. 1.

Targeted disruption of the Prss16 by homologous recombination. (A) A neomycin resistance cassette was inserted between the HindIII and ClaI sites replacing exon 5 (hatched box), which includes the putative active serine residue, through exon 9 and the 5′ end of exon 10. (B) 5′ (upper panel) and 3′ (lower panel) PCR analysis of ES cells. Primers KO3.F and KO3.R were used to screen for 5′ integration of the targeting vector. 3′ integration was confirmed by PCR with primers 5′seq1 and LApcr.R1. M, molecular weight markers (in hundreds); wt, wild type. (C) Genotype analysis of offspring from heterozygous parents of modified Prss16. Tail DNA was used for PCR analyses of the mutant and wild-type alleles with primer pairs KO3.F-KO3.R (lanes 1, 3, 5, 7, 9, 11, 13, 15, 17) and Ex567.F-Ex567.R (lanes 2, 4, 6, 8, 10, 12, 14, 16, 18), respectively. (D) Prss16^−/− mice do not produce Prss16 mRNA. Northern blot of total thymus RNA from Prss16^−/−, Prss16^+/−, and Prss16^+/+ mice probed with a Prss16 cDNA.

FIG. 2.

(A) Western blot analysis with anti-Ii antibody (In-1) reveals no difference in Ii isoforms p41 and p31 and the degradation product p10 in thymic stromal cells of Prss16^−/− mice compared to those of wild-type mice. The genotype of each animal is indicated. (B) MHC class II surface expression on BP-1⁺CD4⁻CD11c⁻ cells. Shown are means and standard errors of the means. Wild-type and heterozygous animals were grouped together (Prss16^+/?), cTEC were identified by gating on low-density thymic cells that were BP-1⁺CD4⁻CD11c⁻, and surface expression of MHC class II was estimated by mean fluorescence intensity (MFI). No significant difference was detected between Prss16^−/− mice and their littermate controls (n = 8 per group). (C) Representative results of flow cytometry of low-density thymic cells stained for BP-1, CD4, CD11c, and MHC class II.

FIG. 3.

The percentages and total numbers of CD4⁺-, CD8⁺-, and CD4⁺CD8⁺-T-cell populations are similar for Prss16^−/− and Prss16^+/+ mice. Thymocytes (A) and splenocytes (B) from Prss16^−/− and Prss16^+/+ littermates were stained with anti-CD4 and anti-CD8 monoclonal antibodies (MAb). The percentage of the total number of T cells in each population is indicated. The total numbers of T cells in the thymus and spleen of Prss16^−/− mice did not differ significantly. (C) The percentage of CD3⁻ thymocytes expressing CD44, CD25, or both did not differ significantly between Prss16^−/− (solid bar) and Prss16^+/+ (hatched bar) littermates. Thymocytes were stained with anti-CD3, anti-CD44, and anti-CD25 MAb. The CD3⁻ population was gated for analysis of CD44 and CD25. The percentage of the total number of T cells in each population is indicated.

FIG. 4.

(A) The percentages of thymic and splenic CD4⁺CD25⁺ T cells were not affected by deletion of Prss16. Splenic and thymic cells of Prss16^+/+ and Prss16^−/− mice were stained with anti-TCRβ, anti-CD4, and anti-CD25. The percentages of TCRβ⁺CD4⁺ cells expressing CD25 are indicated. (B) NK T cells identified as TCRβ⁺DX5⁺ from spleens of Prss16^+/+ and Prss16^−/− mice were analyzed by using three-color FACS analysis. The numbers indicate the percentages of DX5⁺ T cells.

FIG. 5.

Activation of splenocytes by anti-CD3 MAb and concanavalin A from Prss16^−/− mice is not different from that from control littermates. Splenocytes were treated for 48 h with either anti-CD3 MAb or concanavalin A, and proliferation was assessed by an MTT assay. Bars represent the means ± standard deviations of the differences in absorbance at 570 nm between anti-CD3-, concanavalin A-, and PBS-treated cells. Each group of 10 mice was assayed in triplicate.