How alpha beta T cells deal with induced TCR alpha ablation

Abstract

On deletion of the gene encoding the constant region of the T cell antigen receptor (TCR)alpha chain in mature T cells by induced Cre-mediated recombination, the cells lose most of their TCR from the cell surface within 7--10 days, but minute amounts of surface-bound TCR beta chains are retained for long periods of time. In a situation in which cellular influx from the thymus is blocked, TCR-deficient naive T cells decay over time, the decay rates being faster for CD8(+) cells (t(1/2) approximately 16 days) than for CD4(+) cells (t(1/2) approximately 46 days). TCR(+) naïve cells are either maintained (CD8(+)) or decay more slowly (CD4(+); t(1/2) approximately 78 days.) Numbers of TCR-deficient memory T cells decline very slowly (CD8(+) cells; t(1/2) approximately 52 days) or not at all (CD4(+) cells), but at the population level, these cells fail to expand as their TCR(+) counterparts do. Together with earlier data on T cell maintenance in environments lacking appropriate major histocompatibility complex antigens, these data argue against the possibility that spontaneous ligand-independent signaling by the alpha beta TCR contributes significantly to T-cell homeostasis.

Figure 1

Targeting of the TCR Ca locus. (a) Partial restriction map of the TCR Ca locus. Restriction sites for BamHI (B) and HindIII (H) endonuclases are shown. Jα elements and Cα exons are depicted as closed squares and the TCRα enhancer, as a closed circle. External hybridization probes A and B, used to verify homologous recombination of the targeting vector by Southern blot, together with expected sizes of the DNA fragments, are indicated below the line. (b) Targeted TCR Cα locus. LoxP sites, framed closed triangles; neo cassette, dotted square. Introduction of the loxP site in the Jα-Cα intron caused destruction of a BamHI site, which was used to determine homologous recombination and third loxP cointegration by probe A. Probe C was used to exclude random integration of the vector. (c) Conditional Cα allele after deletion of the neo cassette by transient expression of Cre recombinase in targeted ES cells. Small arrows show PCR primers (loxP F and Cα4 R) designed for detection of the loxP-flanked Cα allele. (d) Deletion of TCR Cα genes after induction of Cre expression in vivo.

Figure 2

(A) Decay of TCR from T cells upon conditional Cα deletion. Flow cytometric determination of TCRβ expression on CD4⁺ (Left) and CD8⁺ (Right) peripheral blood T cells. Days after the injection of pI/pC and percentages of T cells with down-regulated TCRβ are indicated. (B) Simultaneous decay of CD3 complex with TCRβ on Cα-deleted T cells. Dot plot represents CD5-gated peripheral blood lymphocytes on day 15 after injection of pI/pC. Cells were analyzed for surface CD3ɛ and TCRβ expression by flow cytometry. (C) Southern blot analysis of TCR⁺ and TCR⁻ T cells. TCR⁺ and TCR⁻ subsets of CD4⁺ and CD8⁺ T cells isolated from the spleen of Cα^fl/− mice 10 days after the injection of pI/pC were sorted, and genomic DNA was analyzed for Cα deletion. BamHI-digested DNA was hybridized with probe A (see Fig. 1A). Bands indicate TCRα^−/− (6.9 kb), Cα-deleted (Cα^Δ, 6.7 kb), and Cα^fl (10.9 kb) alleles.

Figure 3

(A) Persistence of low levels of TCR on the surface of T cells upon Cα deletion. CD3⁺ and CD3⁻ fractions of CD4⁺ and CD8⁺ T cells were analyzed for TCRβ expression by staining with an anti-TCRβ mAb and magnetofluorescent liposomes. Dot plots show gated fractions of CD4⁺ (Left) and CD8⁺ (Right) peripheral blood T cells. Histograms below show expression of TCRβ by liposome staining on gated fractions of T cells. Percentages of TCRβ-positive cells and time of analysis are indicated. (B) Sensitivity of liposome-assisted staining for TCRβ. Sensitivity of liposome staining for TCRβ was tested on CD4⁺ CD8⁺ TCRα^−/− thymocytes. Data are represented as histograms, and percentages of TCRβ⁺ cells are indicated. Staining of thymocytes from RAG1-deficient mice, as a negative control, shows background of the liposome-assisted staining.

Figure 4

Decay of T cells upon TCR Cα deletion. Percentages of CD44^lo (Left) and CD44^hi (Right) CD4⁺ (Upper) and CD8⁺ cells (Lower) in the spleens of the experimental animals at various times after pI/pC injection. Open circles, TCR⁺ cells; closed circles, TCR⁻ cells. Experimental points come from individual animals. Half-lives (t_1/2) were calculated from regression lines (sigma plot program, Ver. 4.0, SPSS, Chicago).

Figure 5

Proliferation of TCR⁻ memory T cells. Cα^fl/−, Mx-Cre, and Cα^fl/− mice were fed with BrdU containing drinking water from day 60 to 67 after pI/pC injection. Controls did not receive BrdU. TCR⁺ and TCR⁻ subsets of CD4⁺ and CD8⁺ T cells were then sorted and stained for CD44 and BrdU. Percentages of cells in various subpopulations are indicated.

Figure 6

Functional properties of TCR⁻ T cells. (A) Lack of proliferation on TCR crosslinking in vitro. FACS-sorted TCR⁺ and TCR⁻ subsets of CD4⁺ and CD8⁺ splenic T cells, isolated on day 10 after pI/pC injection, were labeled with CFDA-SE, incubated in the presence of either anti-CD3ɛ or anti-TCRβ plate-bound mAbs for 72 h, and subsequently analyzed by flow cytometry. (B) Lack of Ca²⁺ mobilization on CD3ɛ crosslinking. Spleen cells were isolated on day 12 after pI/pC injection, loaded with the Ca²⁺-sensitive dye Indo-1, and subsequently stained for CD4 or CD8 and CD3e. Relative intracellular Ca²⁺ concentration was measured on gated CD3ɛ⁺ and CD3ɛ⁻ T cells during the time period of 512 s. (C) Proliferation of TCR⁻ CD8⁺ memory T cells on stimulation with IL-15. Magnetically sorted and CFDA-SE-labeled splenic CD8⁺ T cells were incubated for 72 h in the presence of human recombinant IL-15 at various concentrations, as indicated. Subsequently, the cells were stained for CD8, TCRβ, and CD44 and analyzed by flow cytometry.