Bcl-2 Is Necessary to Counteract Bim and Promote Survival of TCRαβ+CD8αα+ Intraepithelial Lymphocyte Precursors in the Thymus

Abstract

The precursors of TCRαβ⁺CD8αα⁺ intraepithelial lymphocytes (IEL) arise in the thymus through a complex process of agonist selection. We and others have shown that the proapoptotic protein, Bim, is critical to limit the number of thymic IEL precursors (IELp), as loss of Bim at the CD4⁺CD8⁺ double-positive stage of development drastically increases IELp. The factors determining this cell death versus survival decision remain largely unknown. In this study, we used CD4CreBcl2^f/f mice to define the role of the antiapoptotic protein Bcl-2 and CD4CreBcl2^f/fBim^f/f mice to determine the role of Bcl-2 in opposing Bim to promote survival of IELp. First, in wild-type mice, we defined distinct subpopulations within PD-1⁺CD122⁺ IELp, based on their expression of Runx3 and α4β7. Coexpression of α4β7 and Runx3 marked IELp that were most dependent upon Bcl-2 for survival. Importantly, the additional loss of Bim restored Runx3⁺α4β7⁺ IELp, showing that Bcl-2 antagonizes Bim to enable IELp survival. Further, the loss of thymic IELp in CD4CreBcl2^f/f mice also led to a dramatic loss of IEL in the gut, and the additional loss of Bim restored gut IEL. The loss of gut IEL was due to both reduced seeding by IELp from the thymus as well as a requirement for Bcl-2 for peripheral IEL survival. Together, these findings highlight subset-specific and temporal roles for Bcl-2 in driving the survival of TCRαβ⁺CD8αα⁺ IEL and thymic IELp.

Fig 1.. Bcl-2 contributes to the survival of CD5+ TCRβ+ DN thymocytes

(A) Within CD4+ CD8+ thymocytes, triple positive (CD4+ CD8αα+ CD8αβ+) pre-selection precursors of TCRαβ+ CD8αα+ IEL were identified by gating on Thymus Leukemia (TL) antigen tetramer positive cells and CD8β. (B) CD25− CD1dtet− CD4− CD8− DN (top row) and within the DN, post-selected CD5+ TCRβ+ thymocytes (bottom row) were determined. Numbers in representative plots show the percentage and bar graphs show the numbers of each population from Bcl2^f/f (open circle) and CD4CreBcl2^f/f (filled circle) mice. (C) Histograms show the mean fluorescence intensity(MFI) of Bcl-2 and Bim in CD5+ TCRβ+ DN, CD5− TCRβ− DN, DP and CD8+ thymocytes from Bcl2^f/f and CD4CreBcl2^f/f mice. Results are representative of at least three independent experiments with n=3 or more mice per group and show mean ± SD. **p < 0.01, Student’s t test.

Fig 2.. Bcl-2 is critical for the survival of CD122+ thymocytes

(A) Within CD5+ TCRβ+ DN thymocytes, PD-1 and Tbet expressing populations were identified. (B) Within the PD-1+ and Tbet+ cells, CD122 expressing populations were identified. Numbers in representative plots show the percentage and bar graphs show the numbers from Bcl2^f/f (open circle) and CD4CreBcl2^f/f (filled circle) mice. (C) Histograms show Bcl-2 and Bim expression in PD-1+ CD122−, PD-1+ CD122+ and Tbet+ populations in Bcl2^f/f and CD4CreBcl2^f/f mice. Results are representative of at least four independent experiments with n=3 or more mice per group and show mean ± SD. **p < 0.01, Student’s t test.

Fig 3.. Runx3 expression marks a mature IELp sub-population among PD-1+ CD122+ thymocytes that critically depends on Bcl-2 for survival

(A) Flow cytometric identification of Runx3− α4β7− , Runx3− α4β7+, Runx3+ α4β7− and Runx3+ α4β7+ sub-populations within PD0-1+ CD122+ CD44^lo post-selected DN thymocytes. The bar graphs show the numbers of each population from Bcl2^f/f (open circle) and CD4CreBcl2^f/f (filled circle) mice. (B) Representative dot plots and bar graphs show the percentage of CD69+ cells among sub-populations of PD-1+ CD122+ CD44^lo thymocytes in WT mice (C) Bar graphs show the MFI of Bcl-2 and Bim in the sub-populations of PD-1+ CD122+CD44^lo thymocytes from WT mice in comparison to PD-1+ CD122− cells. Results are representative of 3 independent experiments with n=3 or more mice per group and show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001 Student’s t test.

Fig 4.. Bcl-2 antagonizes Bim to maintain survival of CD122+ IELp

Dot plots show frequencies and bar graphs show thymocyte numbers from WT (open circle), CD4Cre+Bcl2^f/f (filled circle), and CD4CreBim^f/f (open triangle) and CD4CreBim^f/f Bcl2^f/f (filled square) mice of (A) DN (CD25− CD1tet− CD4− CD8−) (Row 1) , CD5+ TCRβ+ among DN (Row 2), PD-1+ and Tbet+ among CD5+ TCRβ+ DN (Row 3) , and CD122+ and CD122− among the PD-1+ (Row 4) (B) Runx3− α4β7− , Runx3− α4β7+ , Runx3+ α4β7− and Runx3+ α4β7+ cells within PD-1+ CD122+ CD44^lo thymocytes (C) Bar graph compares MFI of Runx3 in the Runx3+ α4β7+ sub-population in WT (open circle), CD4CreBcl2^f/f (filled circle), and CD4CreBim^f/f (open triangle) mice. (D) Bar graphs compare MFI of Bcl-2 in Runx3− α4β7− , Runx3− α4β7+ , Runx3+ α4β7− and Runx3+ α4β7+ sub-populations between WT (open circle) and CD4CreBim^f/f (filled square) mice and of Bim between WT (open circle) and CD4CreBcl2^f/f (filled circle) mice. Results are representative of at least 3 independent experiments with n=3 or more mice per group and show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001 Student’s t test.

Fig 5.. Thymic deletion of Bcl-2 leads to a profound loss of TCRαβ+ CD8αα+ IEL

(A)Representative dot plots indicate frequency of gut IEL populations ( gated from live, CD45.2+ cells) and show TCRβ+ and TCRδ+ IEL(top row), CD8α and CD4 expression among TCRβ+ IEL(middle row) and CD8β expression among the CD8α+ IEL ( bottom row) .The bar graph shows the numbers of TCRαβ+ CD8αα+, TCRαβ+ CD8αβ+ and TCRαβ+ CD4+ IEL from 8 week old Bcl2^f/f (open circle) and CD4CreBcl2^f/f (filled circle) mice. (B) Histograms show Bcl-2 expression in TCRαβ+ CD8αα+ IEL (left column) and TCRαβ+ CD8αβ+ IEL (right column) from 3 week old and 5 week old WT (solid no fill) and CD4CreBcl2^f/f (solid grey fill) mice and 5 week old WT (solid no fill) and CD4CreBcl2^f/f Bim^f/f (dashed light grey fill) mice. (C) Bar graphs show numbers of IEL in WT (open circle) and CD4CreBcl2^f/f (filled circle) at 3 weeks of age and CD4CreBim^f/f (open triangle) and CD4CreBim^f/f Bcl2^f/f (filled square) at 5 weeks of age. D) Bar graph shows the frequency of Ki-(A) 67+ cells among TCRαβ+ CD8αα+, TCRαβ+ CD8αβ+ and TCRαβ+ CD4+ IEL from 3 week old Bcl2^f/f mice (open circles) and CD4Cre+Bcl2^f/f mice (filled circles). Results are representative of at least 3 independent experiments with n=3 or more mice per group and show mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001 Student’s t test.