Negative selection, not receptor editing, is a physiological response of autoreactive thymocytes

Abstract

Antigen receptor editing-a process of secondary rearrangements of antigen receptor genes in autoreactive lymphocytes-is a well-established tolerance mechanism in B cells, whereas its role in T cells remains controversial. Here, we investigated this issue using a novel Tcra knock-in locus, which ensured appropriate timing of TCRα expression and allowed secondary rearrangements. Under these conditions the only response to self-antigen that could be unambiguously identified was negative selection of CD4/CD8 double positive thymocytes. No evidence could be obtained for antigen-induced TCR editing, whereas replacement of the transgenic TCRα chain by ongoing gene rearrangement occurred in some cells irrespective of the presence or absence of self-antigen.

Figure 1.

Corrected timing of TCRα expression and normal gross thymic phenotype of HY-STOP mice. (A) Targeting strategy for generation of the Tcra^HY-STOP locus. Original HY-I knock-in locus (top), targeting vector (center), and targeted locus with a STOP cassette are shown. HY VαJα, recombined VαJα element of HY TCR; Neo, neomycin resistance gene; frt, frt sites; pA, polyadenylation signal; STOP, transcriptional STOP cassette; loxP, leftover loxP site in HY-I locus; loxP2272, alternative loxP sites; TK, thymidine kinase gene; Amp, ampicillin resistance gene. (B) Expression of surface TCRβ and HYα on CD4⁻CD8⁻ (DN), CD4⁺CD8⁺ (DP), and CD4⁻CD8⁺ (CD8SP) thymocytes from the HY-I (Tcra^HY-I/HY-I HYβ) and HY-STOP (Tcra^HY-STOP/− HYβ Rorγt-Cre) female mice. Representative results of at least three independent experiments are shown. (C) Total thymocytes numbers and numbers of TCR⁻ DP thymocytes in male and female HY-I and HY-STOP mice. *, P < 0.5; **, P < 0.01 (n = 3-5); student’s t test. Error bars represent the SD. Representative results of two independent experiments are shown. (D) Comparison of CD4/CD8 profiles of HY-I and HY-STOP thymocytes from male and female mice. Representative results of at least three independent experiments are shown.

Figure 2.

HY-STOP Tcra locus is functional and can undergo antigen-independent secondary rearrangements. (A) Female HY-STOP thymocytes were stained for surface TCRβ, HYα, and Vα2 or Vα3.2. Note the presence of TCRβ⁺HYα⁻ cells (left), a fraction of which can be stained with Vα antibodies (center), and the lack of binding of Vα antibodies to TCRβ⁺HYα⁺ cells (right). Representative results of two independent experiments are shown. (B) Female HY-STOP thymocytes were stained for surface TCRβ, HYα, TCRβ8.1/8.2, CD4, and CD8α. Staining for TCRβ and TCRβ8.1/8.2 on HYα⁻ cells (left) and expression of CD4 and CD8α by TCRβ⁺HYα⁻ cells (right) is shown. Representative staining for one of three analyzed animals is shown.

Figure 3.

Presence of self-antigen does not enhance secondary rearrangements. (A) Schematic representation of the predicted effects of deletion and TCR-editing scenarios on numbers of clonotype-positive and -negative thymocytes. (B) Expression of surface TCRβ and HYα on DP and CD8SP thymocytes from HY-STOP male and female BM chimeras. Representative results of at least five independent experiments are shown. (C) Absolute numbers (left) and percentage from total thymocytes (right) of TCRβ⁺HYα⁺ and TCRβ⁺HYα⁻ DP cells in male and female HY-STOP BM chimeras. **, P < 0.01; NS, not significant (P > 0.05); student’s t test; n = 5. Error bars represent the SD. Representative results of two independent experiments are shown. (D) Expression of Rag1 and Rag2 was analyzed by TaqMan qPCR in sorted TCRβ⁻ and TCRβ⁺HYα⁺ DP thymocytes from HY-STOP male and female BM chimeras. Mean relative expression normalized against Gapdh expression is shown. Error bars represent SD (individual mice; n = 4 for each group from two independent experiments). ***, P < 0.001; **, P < 0.01; student’s t test; NS, not significant (P > 0.05).

Figure 4.

HYα⁺ cells in male HY-STOP mice are negatively selected. (A) Expression of CD4 and CD8 co-receptors by male and female HYα⁺ thymocytes. Representative results of at least five independent experiments are shown. Note co-receptor dulling and lack of CD4⁻CD8⁺ cells among male clonotype-positive cells. (B) Expression of negative selection–associated markers PD-1 and Helios by total HYα⁺ DP thymocytes from male and female HY-STOP mice and by DP^dull cells from male mice. Representative results of three independent experiments are shown. (C) Detection of cleaved caspase-3 in male and female HYα⁺ thymocytes from HY-I (top) and HY-STOP (bottom) mice. Representative results of two independent experiments are shown.