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. 2020 Mar 3;9:e52539. doi: 10.7554/eLife.52539

Figure 3. Establishment of Pmel-1 x SLAMF6 -/- mice as a source of SLAMF6-KO antigen-specific lymphocytes.

(A) SLAMF6 and Vβ13 expression in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes measured by flow cytometry. (B) Percent CD8+, CD4, and CD19 cells in spleens from Pmel-1 or Pmel-1 x SLAMF6 -/- untreated mice. (C) Pmel-1, and Pmel-1 x SLAMF6 -/- CD8+ untreated splenocytes were stained with anti-CD44 and anti-CD62L. One representative experiment is shown. (D) Percent CD8+ cells in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes after 7 days of in vitro activation with gp10025-33 peptide and IL-2 (30 IU/ml). (E) Flow cytometry for activation markers (CD25, CD69, CD137) in Pmel-1 or Pmel-1 x SLAMF6 -/- splenocytes after 3 days of in vitro activation, as in (D). Median fluorescence intensity (MFI) is shown. (F) Expression of PD-1 in Pmel-1 or Pmel-1 x SLAMF6 -/- CD8+ T cells after 7 days of in vitro activation, as in (D). Median fluorescence intensity (MFI) is shown. (G, H) After 7 days of activation, Pmel-1 and Pmel-1 x SLAMF6 -/- CD8+ T cells were stained with anti-CD44 and anti-CD62L. CD8+ subpopulations were defined for each mouse strain. (G) One representative experiment and (H) summary of subpopulations identified by flow cytometry in five experiments is shown. EM, effector memory, CM, central memory. Student t-test. *, p<0.05, **, p<0.01, ***, p<0.001.

Figure 3.

Figure 3—figure supplement 1. Characterization of Pmel-1 x SLAMF6 -/- mice.

Figure 3—figure supplement 1.

(A) Immunohistochemistry staining of Pmel-1 and Pmel-1 x SLAMF6 -/- spleen sections using anti CD4 and anti-CD8+ antibodies (X10 magnification). (B) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes were labeled with CFSE and activated; at the indicated time points the cells were stained for CD8+ expression, and CFSE level gated on the CD8+ population was measured using flow cytometry. (C) Pmel-1 and Pmel-1 x SLAMF6 -/- splenocytes activated for 7 days followed by 7 days maintenance with IL-2 (30 IU/ml) or without its addition. Percentage apoptotic and dead cells was measured by PI-Annexin V. Summary of two experiments shown. No Tx, no treatment. (D) After 7 days of activation, Pmel-1 and Pmel-1 x SLAMF6 -/- CD8+ T cells were stained with antibodies against SLAM family receptors. The expression level of each receptor in CD8+ cells is presented.