T cell development in mice lacking all T cell receptor zeta family members (Zeta, eta, and FcepsilonRIgamma)

Abstract

The zeta family includes zeta, eta, and FcepsilonRIgamma (Fcgamma). Dimers of the zeta family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking zeta/eta chains, T cell development is impaired, yet low numbers of CD4+ and CD8+ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a zeta family dimer can promote T cell maturation, or that in the absence of zeta/eta, Fcgamma serves as a subunit in TCR complexes. To elucidate the role of zeta family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only zeta/eta or Fcgamma. The data reveal that surface complexes that are expressed in the absence of zeta family dimers are capable of transducing signals required for alpha/beta-T cell development. Strikingly, T cells generated in both zeta/eta-/- and zeta/eta-/--Fcgamma-/- mice exhibit a memory phenotype and elaborate interferon gamma. Finally, examination of different T cell populations reveals that zeta/eta and Fcgamma have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of zeta family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.

Figure 1

Phenotypic analysis of thymocytes and T cells from ζ/η^+/+–Fcγ^+/+, ζ/η^−/−–Fcγ^−/− , ζ/η^−/−–Fcγ^+/+, and ζ/η^+/+–Fcγ^−/− mice. (A) CD4, CD8, and CD3 expression on total thymocytes. (B) CD4 versus CD8 expression on ungated splenocytes. CD3 expression is shown on CD4⁺ and CD8⁺ gated splenocytes (CD4⁺/CD8⁺). (C) CD25 versus CD44 expression on gated (CD4⁻CD8⁻CD3⁻B220⁻) thymocytes. Immunofluorescence and flow cytometric analysis was performed with thymocytes and splenocytes from young adult (4–8 wk-old) mice. Shaded areas represent staining with experimental antibodies and solid lines represent staining with negative control antibody. Numbers in quadrants of dual parameter histograms represent the percentage of cells contained within that quadrant. (D) Induction of CD4⁺CD8⁺ thymocytes in RAG1^−/−, ζ/η^−/− and in RAG1^−/−, ζ/η^−/−–Fcγ^−/− mice by anti-CD3-ε antibodies. 6–12-wk-old mice were injected intraperitoneally with 200 μl PBS containing 75 μg of affinity purified CD3-ε specific antibody 145-2C11. Control animals were injected with 200 μl PBS. 6 d after injection, thymocytes were counted and analyzed by flow cytometry for expression of CD4 and CD8. Results shown are representative of three separate experiments for each genotype.

Figure 1

Phenotypic analysis of thymocytes and T cells from ζ/η^+/+–Fcγ^+/+, ζ/η^−/−–Fcγ^−/− , ζ/η^−/−–Fcγ^+/+, and ζ/η^+/+–Fcγ^−/− mice. (A) CD4, CD8, and CD3 expression on total thymocytes. (B) CD4 versus CD8 expression on ungated splenocytes. CD3 expression is shown on CD4⁺ and CD8⁺ gated splenocytes (CD4⁺/CD8⁺). (C) CD25 versus CD44 expression on gated (CD4⁻CD8⁻CD3⁻B220⁻) thymocytes. Immunofluorescence and flow cytometric analysis was performed with thymocytes and splenocytes from young adult (4–8 wk-old) mice. Shaded areas represent staining with experimental antibodies and solid lines represent staining with negative control antibody. Numbers in quadrants of dual parameter histograms represent the percentage of cells contained within that quadrant. (D) Induction of CD4⁺CD8⁺ thymocytes in RAG1^−/−, ζ/η^−/− and in RAG1^−/−, ζ/η^−/−–Fcγ^−/− mice by anti-CD3-ε antibodies. 6–12-wk-old mice were injected intraperitoneally with 200 μl PBS containing 75 μg of affinity purified CD3-ε specific antibody 145-2C11. Control animals were injected with 200 μl PBS. 6 d after injection, thymocytes were counted and analyzed by flow cytometry for expression of CD4 and CD8. Results shown are representative of three separate experiments for each genotype.

Figure 1

Phenotypic analysis of thymocytes and T cells from ζ/η^+/+–Fcγ^+/+, ζ/η^−/−–Fcγ^−/− , ζ/η^−/−–Fcγ^+/+, and ζ/η^+/+–Fcγ^−/− mice. (A) CD4, CD8, and CD3 expression on total thymocytes. (B) CD4 versus CD8 expression on ungated splenocytes. CD3 expression is shown on CD4⁺ and CD8⁺ gated splenocytes (CD4⁺/CD8⁺). (C) CD25 versus CD44 expression on gated (CD4⁻CD8⁻CD3⁻B220⁻) thymocytes. Immunofluorescence and flow cytometric analysis was performed with thymocytes and splenocytes from young adult (4–8 wk-old) mice. Shaded areas represent staining with experimental antibodies and solid lines represent staining with negative control antibody. Numbers in quadrants of dual parameter histograms represent the percentage of cells contained within that quadrant. (D) Induction of CD4⁺CD8⁺ thymocytes in RAG1^−/−, ζ/η^−/− and in RAG1^−/−, ζ/η^−/−–Fcγ^−/− mice by anti-CD3-ε antibodies. 6–12-wk-old mice were injected intraperitoneally with 200 μl PBS containing 75 μg of affinity purified CD3-ε specific antibody 145-2C11. Control animals were injected with 200 μl PBS. 6 d after injection, thymocytes were counted and analyzed by flow cytometry for expression of CD4 and CD8. Results shown are representative of three separate experiments for each genotype.

Figure 1

Phenotypic analysis of thymocytes and T cells from ζ/η^+/+–Fcγ^+/+, ζ/η^−/−–Fcγ^−/− , ζ/η^−/−–Fcγ^+/+, and ζ/η^+/+–Fcγ^−/− mice. (A) CD4, CD8, and CD3 expression on total thymocytes. (B) CD4 versus CD8 expression on ungated splenocytes. CD3 expression is shown on CD4⁺ and CD8⁺ gated splenocytes (CD4⁺/CD8⁺). (C) CD25 versus CD44 expression on gated (CD4⁻CD8⁻CD3⁻B220⁻) thymocytes. Immunofluorescence and flow cytometric analysis was performed with thymocytes and splenocytes from young adult (4–8 wk-old) mice. Shaded areas represent staining with experimental antibodies and solid lines represent staining with negative control antibody. Numbers in quadrants of dual parameter histograms represent the percentage of cells contained within that quadrant. (D) Induction of CD4⁺CD8⁺ thymocytes in RAG1^−/−, ζ/η^−/− and in RAG1^−/−, ζ/η^−/−–Fcγ^−/− mice by anti-CD3-ε antibodies. 6–12-wk-old mice were injected intraperitoneally with 200 μl PBS containing 75 μg of affinity purified CD3-ε specific antibody 145-2C11. Control animals were injected with 200 μl PBS. 6 d after injection, thymocytes were counted and analyzed by flow cytometry for expression of CD4 and CD8. Results shown are representative of three separate experiments for each genotype.

Figure 2

CD69 and CD5 upregulation on DP thymocytes in response to TCR engagement. DP thymocytes were purified and stimulated for 12–16 h on plates coated with either PBS or anti–TCR-β. Cells were then stained with anti-CD69 or anti-CD5 and analyzed by FCM. Shaded areas depict cells stained with anti-CD69 or anti-CD5 after anti–TCR-β stimulation. Solid lines depict cells stained with anti-CD69 or anti-CD5 after incubation in media without antibody stimulation.

Figure 3

Surface phenotype of peripheral T cells from ζ/η^−/−– Fcγ^−/− mice. Activation-memory phenotype of splenic T cells. Cells were stained with anti-CD4-PE versus anti-CD44-FITC, anti-CD62L-FITC, anti-CD2-FITC, or anti-CD5-FITC. Single color histograms show staining on CD4⁺ T cells. Data were collected on 5 × 10³ gated cells. Shaded areas represent staining with experimental antibodies and solid lines depict staining with negative control antibodies.

Figure 4

Semiquantitative RT-PCR for detection of IFN-γ, IL-2, or IL-4 mRNA. RNA obtained from unstimulated purified splenic CD4⁺ and CD8⁺ T cells (ex vivo) or after 18 h of stimulation with PMA + ionomycin was reverse transcribed and amplified with oligonucleotide primers specific for IL-2, IL-4, and IFN-γ. Reactions were standarized by performing PCR with oligonucleotides corresponding to cyclophilin and CD3-ε, whose mRNAs should be equivalent in purified T cell populations. +, indicates homozygosity for the wild-type ζ/η or Fcγ alleles; − indicates homozygosity for the mutant ζ/η or Fcγ alleles, as indicated.

Figure 5

i-IEL development in ζ/η^−/−–Fcγ^−/− mice i-IELs were prepared from mice as described (13) and three-color FCM was performed. For internal staining, cells were first stained with anti-CD4 and anti– CD8-β externally, then treated with intracellular staining buffer followed by staining with anti-CD3, anti–TCR-β, or anti–TCR-δ mAbs. Data depict two-color analysis of CD3 versus TCR-β or CD3 versus TCR-δ on software-gated CD4⁻ CD8-β⁻ cells. Numbers reflect the percentage of gated CD4⁻CD8-β⁻ cells in that quadrant.

Figure 6

Phenotype of DETC and DN NK1.1⁺ thymocytes from ζ/η^+/+–Fcγ^+/+, ζ/η^−/−–Fcγ^+/+, ζ/η^+/+–Fcγ^−/−, and ζ/η^−/−–Fcγ^−/− mice. (A) DETCs were purified as described in Materials and Methods and stained with antibodies directed against TCR-γδ and CD3-ε or Thy 1 and CD45. (B) Expression of surface TCR on NK1.1⁺ thymocytes from ζ/η^+/+–Fcγ^+/+, ζ/η^−/−–Fcγ^+/+ and ζ/η^−/−–Fcγ^−/− mice. Shown are two-color plots of NK1.1 versus. α/β-TCR or γ/δ-TCR on gated (CD24⁻) thymocytes. Numbers in quadrants refer to percent of NK1.1⁺ thymocytes that express TCR (α/β-TCR or γ/δ-TCR). Single-color plots show expression of α/β-TCR on gated NK1.1⁺ thymocytes. Shaded areas in single color histograms represent staining with negative control antibodies.

Figure 6

Phenotype of DETC and DN NK1.1⁺ thymocytes from ζ/η^+/+–Fcγ^+/+, ζ/η^−/−–Fcγ^+/+, ζ/η^+/+–Fcγ^−/−, and ζ/η^−/−–Fcγ^−/− mice. (A) DETCs were purified as described in Materials and Methods and stained with antibodies directed against TCR-γδ and CD3-ε or Thy 1 and CD45. (B) Expression of surface TCR on NK1.1⁺ thymocytes from ζ/η^+/+–Fcγ^+/+, ζ/η^−/−–Fcγ^+/+ and ζ/η^−/−–Fcγ^−/− mice. Shown are two-color plots of NK1.1 versus. α/β-TCR or γ/δ-TCR on gated (CD24⁻) thymocytes. Numbers in quadrants refer to percent of NK1.1⁺ thymocytes that express TCR (α/β-TCR or γ/δ-TCR). Single-color plots show expression of α/β-TCR on gated NK1.1⁺ thymocytes. Shaded areas in single color histograms represent staining with negative control antibodies.