Antagonist peptide selects thymocytes expressing a class II major histocompatibility complex-restricted T cell receptor into the CD8 lineage

Abstract

CD4/CD8 lineage decision is an important event during T cell maturation in the thymus. CD8 T cell differentiation usually requires corecognition of major histocompatibility complex (MHC) class I by the T cell receptor (TCR) and CD8, whereas CD4 T cells differentiate as a consequence of MHC class II recognition by the TCR and CD4. The involvement of specific peptides in the selection of T cells expressing a particular TCR could be demonstrated so far for the CD8 lineage only. We used mice transgenic for an MHC class II-restricted TCR to investigate the role of antagonistic peptides in CD4 T cell differentiation. Interestingly, antagonists blocked the development of CD4(+) cells that normally differentiate in thymus organ culture from those mice, and they induced the generation of CD8(+) cells in thymus organ culture from mice impaired in CD4(+) cell development (invariant chain-deficient mice). These results are in line with recent observations that antagonistic signals direct differentiation into the CD8 lineage, regardless of MHC specificity.

Figure 1

Peptides 113I and 113V antagonize the activation of A18 T cells. IL-2 response of A18 TCR transgenic spleen cells in response to dendritic cells prepulsed with 10 nM A18 agonist peptide, and then cultured in the presence of peptide 113I (▪), peptide 113V (♦), and HEL peptide 1–18 (•) at indicated concentrations. The figure shows counts per minute of [³H]thymidine incorporated into CTLL cells.

Figure 2

Generation of MHC class II-restricted CD8⁺ cells in NTOC in the presence of antagonist peptide. Thymocytes from A18 Rag-1^−/− newborns on the Ii^−/− background (A) or Ii^+/− background (B) were analyzed for their expression of CD4, CD8, TCR, and HSA 7 d after organ culture in the absence of peptide (medium) or in the presence of 1 or 10 μM antagonist peptide 113V. Percentages of CD4 and CD8 single positive cells are given in each dot plot. Expression of Vβ8.3 and HSA is shown on gated CD4 single positive cells and gated CD8 single positives, as indicated. Gray-filled curves in each histogram represent expression on thymocytes from NTOC in medium alone for comparison.

Figure 3

Thymuses from A18 Rag1^−/− newborns on the Ii^−/− background were cultured with 10 μM antagonist peptide 113V in the presence of 50 μg/ml anti–class II antibody 14.4.4 (right) or mouse IgG as control (left). Percentages of CD4 and CD8α are given in each dot plot. The histogram below shows expression of CD8β on cells gated for CD8α expression to illustrate coexpression of both CD8 chains.

Figure 4

Increased absolute numbers of CD8 single positive cells after NTOC in the presence of antagonist peptide. Thymic lobes from A18 Rag-1^−/− Ii^−/− newborn mice were cultured in medium alone or in medium containing 1 or 10 μM antagonist peptide 113V for 7 d. Thymocytes were counted, stained for the expression of CD4 and CD8, and analyzed by flow cytometry. Absolute numbers of CD8 single positive cells per lobe of five lobes from four independent experiments are shown.

Figure 5

CD8⁺ cells recovered from NTOC cultured in the presence of antagonist peptide are functionally mature. Thymocytes were recovered from A18 Rag-1^−/− Ii^−/− neonatal thymic lobes after 7 d culture in medium alone or in medium containing different concentrations of antagonist peptide 113V as indicated. Total thymocytes (○), thymocytes depleted of CD4⁺ cells (▪), or thymocytes depleted of CD8⁺ cells (▴) were cocultured with dendritic cells in the presence of 1 μM C5 peptide and the supernatants were assayed in serial dilutions for their content of IL-2. The figure shows the mean counts per minute (of duplicate experiments) of incorporated [³H]thymidine by CTLL cells. Cultures without C5 peptide did not contain IL-2 (not shown).