Thymocyte development is normal in CTLA-4-deficient mice

Abstract

Recent studies indicate that CTLA-4 interaction with B7 ligands transduces an inhibitory signal to T lymphocytes. Mice homozygous for a null mutation in CTLA-4 have provided the most dramatic example of the functional importance of CTLA-4 in vivo. These animals develop a fatal lymphoproliferative disorder and were reported to have an increase in CD4(+) and CD8(+) thymocytes and CD4(-)CD8(-) thymocytes, and a decrease in CD4(+)CD8(+) thymocytes. Based on these observations, it was proposed that CTLA-4 is necessary for normal thymocyte development. In this study, CTLA-4-deficient mice carrying an insertional mutation into exon 3 of the ctla-4 gene were generated. Although these mice display a lymphoproliferative disorder similar to previous reports, there was no alteration in the thymocyte profiles when the parathymic lymph nodes were excluded from the thymi. Further, thymocyte development was normal throughout ontogeny and in neonates, and there was no increase in thymocyte production. Finally, T cell antigen receptor signaling, as assessed by proximal and distal events, was not altered in thymocytes from CTLA-4(-/-) animals. Collectively, these results clearly demonstrate that the abnormal T cell expansion in the CTLA-4-deficient mice is not due to altered thymocyte development and suggest that the apparent altered thymic phenotype previously described was due to the inclusion of parathymic lymph nodes and, in visibly ill animals, to the infiltration of the thymus by activated peripheral T cells. Thus it appears that CTLA-4 is primarily involved in the regulation of peripheral T cell activation.

Figure 1

Apparent thymocyte phenotype in CTLA-4^−/− animals. FACS analysis of all tissue initially believed to be thymus from 18-day-old CTLA-4^−/− and littermate control mice. (a) Total thymocytes. (b) DN thymocytes.

Figure 2

Enlarged parathymic lymph nodes skews the analysis of thymocytes. (A) Parathymic LN uptake of India ink in wild-type (Upper) and CTLA-4^−/− (Lower) mice. (B) FACS analysis of the thymus proper that excludes versus the parathymic LNs that take up the ink was performed. Representative data is shown for 16-day-old CTLA-4^−/− and littermate control mice. The DP population in the parathymic LN is due to thymocytes.

Figure 3

Thymic development in CTLA-4^−/− mice is normal during ontogeny and in newborns. Single cell suspensions were prepared from the thymi and examined by FACS analysis. (A) Fetal thymocytes at E16.5 and E18.5. The expression of αβTCR and γδTCR on the total thymocytes is shown. (B) Thymocytes from 11-day-old animals were stained with antibodies to CD4, CD8, and the indicated cell surface markers. The expression on (a) total thymocytes and (b) DP thymocytes is shown. The number of embryos/animals analyzed at each time point were: E14.5 +/+ n = 2, +/− n = 3, −/− n = 4 (data not shown); E16.5 +/+ n = 1, +/− n = 5, −/− n = 4; E18.5 +/+ n = 2, +/− n = 3, −/− n = 4; D11 +/+ and +/− n = 9, −/− n = 8.

Figure 4

The absence of CTLA-4 does not alter thymocyte cell division. (A) Percentage of BrdU⁺ cells in the DP, total H57^hi thymocytes, and SP (CD4SP >96% αβTCR^hi; CD8SP 45% αβTCR^hi) thymocytes in 11-day-old CTLA-4^−/− mice and littermate controls. (B) Percentage of BrdU⁺ splenic αβTCR⁺ T cells in these animals.

Figure 5

Calcium flux. Thymocytes were loaded with Indo-1 and incubated with anti-CD4-PE and anti-CD8-TC and anti-CD3 (500A2) or 560 antibodies. The ratio of Indo-1-chelated intracellular calcium versus free calcium was measured over time in CTLA-4^−/− and littermate control (A) DP thymocytes and (B) CD4 SP thymocytes from 11-day-old animals.