Fig 1.
T cells from huCD3εHET and huCD3εHOM mice express OKT3 binding site.
(A) Amino acid alignment of corresponding human and mouse CD3e exons. Arrows indicate residues that have been shown to be necessary for OKT3 binding [17]. Mouse exon 5 was replaced with a humanized exon 5 (top line) to generate huCD3εHET or huCD3εHOM mice. (B) Representative flow cytometry plots of mouse splenocytes (gated on scatter profile, PI exclusion, and CD5) using antibodies specific for human (OKT3) or mouse (145-2C11) CD3ε, comparing WT, huCD3εHET, and huCD3εHOM mice. (C) Absolute T cell numbers across each genotype. n = 5–13, p<0.05. (D-F) Representative flow cytometry plots showing the frequency of CD4+ and CD8+ T cells (D), summarized frequencies (E), and absolute cell numbers (F) obtained from spleens of 6-8-week old mice from each genotype. Cells are gated on scatter profile and PI exclusion. (G) CD4:CD8 T cell number ratio from spleens. n = 13–15, *p<0.05.
Fig 2.
HuCD3εHOM, but not huCD3εHET mice, show impaired thymocyte development.
(A) Representative histological staining (H&E) of thymi from WT, huCD3εHET, and huCD3εHOM mice. (B) Flow cytometry plots of murine thymocytes (gated on scatter profile and B220-) comparing CD8+ x CD4+ thymocytes from each genotype. (C-D) Graphs showing frequencies (C) and absolute cell numbers (D) from (B). (E) Cell number ratio of CD4+CD8- to CD4-CD8+ thymocytes observed from each genotype. (F) Flow cytometry plots of murine thymocytes comparing CD25+ x CD44+ thymocytes. Cells are gated on scatter profile and B220-. (G-H) Graphs showing frequencies (G) and absolute cell numbers (H) from (F). n = 3–5, *p<0.05.
Fig 3.
HuCD3εHET peripheral T cells show normal phenotype.
(A) Representative flow cytometry plots of mouse splenocytes comparing CD62L+ x CD44+ expression on CD5+CD4+ (top row) or CD5+CD8+ (bottom row) WT, huCD3εHET and huCD3εHOM T cells. (B-E) Graphs summarizing the frequencies and absolute cell numbers of CD62L+CD44- CD4+ or CD8+ T cells (B, D) and CD44+ CD4+ or CD8+ T cells (C, E) from each genetic background. (F) Graphical summary of CD69 expression on CD4+ or CD8+ T cells from naïve mice from each genotype. (G) Representative flow cytometry plots of apoptosis as measured by Ann-V x 7-AAD in fresh T cells of naïve mice. (H) Graphical summary of (G). n = 5–6, *p<0.05.
Fig 4.
HuCD3ε-expressing T cells respond to anti-human CD3 binding.
(A) Splenocytes from WT and huCD3εHET were cultured in vitro for 24hrs with either anti-human (OKT3) or anti-murine (145-2C11) CD3, their respective isotype controls IgG2a or Armenian Hamster (AH), or anti-mouse TCRβ antibodies. CD69 expression was measured by flow cytometry. (B) Geometric mean fluorescence intensity of murine CD3ε as detected by flow cytometry on CD4+ and CD8+ T cells using 145-2C11 antibody. n = 6, *p<0.05. (C) Mice were administered an i.p. injection of either PBS, anti-mouse CD3 (clone 145-2C11), or anti-human CD3 (clone OKT3). Blood was collected 24hrs post-injection. CD5+ T cell numbers in peripheral blood were determined using CBC and flow cytometry. Individual animals are shown with group mean. n = 5, *p<0.05.
Fig 5.
HuCD3ε-bearing T cells develop functional immune responses.
(A) Splenocytes from 3–4 month old mice from WT or huCD3εHET mice were injected i.v. into 8–9 week old, 800 rads irradiated, B6D2 mice and graft-vs-host disease developed in vivo after a week post-injection. Percent body weight change is shown (A). n = 3–7, *p<0.05. (B-C) 2–4 month-old mice WT and huCD3εHET were immunized against ovalbumin or NP-CGG and administered a booster shot on day 14. Antibody titers were measured on day 10-post immunization (primary response) (B) or seven days following booster shot (secondary response) (C) for each antigen. n = 10, *p<0.05.