CD4+ T cell division in irradiated mice requires peptides distinct from those responsible for thymic selection

Abstract

We investigated the mechanism by which alpha/beta T cells expand upon transfer to T cell-deficient host mice by injecting carboxyfluorescein diacetate succinimidyl ester-labeled T cells into mice depleted of T cells by sublethal irradiation. We found that CD4+ T cells divided when transferred to irradiated hosts and that the division of more than half of these cells required class II expression. However, division of transferred CD4+ T cells did not occur in irradiated hosts that expressed class II molecules occupied solely by the peptide responsible for thymic selection, indicating that peptides distinct from those involved in thymic selection cause the division of CD4+ T cells in irradiated mice. These data establish that class II-bound peptides control the expansion of CD4+ T cells transferred to T cell-deficient hosts and suggest that the same peptides contribute to the maintenance of T cell numbers in normal mice.

Figure 1

CD4⁺ T cells divide after transfer to irradiated hosts. Purified, CFSE-labeled B6.PL (Thy1.1) T cells were transferred to normal or 450-rad irradiated B6 (Thy1.2) mice. Each host received 1.8 × 10⁶ B6.PL CD4⁺ T cells. 7 d later lymph node and spleen cells were stained with PE anti-Th1.1, CyC anti-CD8, and allophycoerythrin anti-CD4. Histograms show the intensity of CFSE fluorescence of CD4⁺Thy1.1⁺ or CD8⁺Thy1.1⁺ cells. Total CD4⁺Thy1.1⁺ cells recovered from the spleen and lymph nodes of each nonirradiated or irradiated host were 2.4 × 10⁵ and 2.7 × 10⁵, respectively.

Figure 2

CD4⁺ T cells divide rapidly after transfer to untreated TCR-βKO (left) or RAG2KO (right) mice. Sublethally irradiated TCR-βKO or RAG2KO mice were injected with 3 × 10⁶ CFSE-labeled B6.PL (left) or BALB/c (right) T cells. 7 d later lymph node and spleen cells were removed for analysis. Cells from TCR-βKO recipients were stained with PE anti-Th1.1, CyC anti-CD8, and allophycoerythrin anti-CD4, and cells from RAG2KO recipients were stained with PE anti-CD4 and CyC anti–TCR-β. The data shown represent CD4⁺Thy1.1⁺ lymph node cells from TCR-βKO recipients and CD4⁺TCR-β¹ lymph node cells from RAG2KO recipients.

Figure 5

Transgenic DO T cells do not divide in irradiated mice that support thymic selection of the DO TCR. DO TCR⁺RAGKO BALB/c, CFSE-labeled cells were transferred to sublethally irradiated BALB/c mice. 24 h later, mice were injected intravenously with soluble OVA peptide as indicated on the figure. 7 d later, cells were stained with PE anti-CD4 and CyC KJ1 (anti-DO idiotype) and analyzed. Data shown are CD4⁺KJ1⁺ lymph node cells.

Figure 3

Much of the proliferation of CD4⁺ T cells in irradiated hosts requires MHC class II expression. 5 × 10⁶ B6.PL CFSE-labeled T cells (2.1 × 10⁶ CD4⁺ T cells) were transferred to 450-rad sublethally irradiated B6 or MHCIIKO mice. 7 d later lymph node and spleen cells were stained and analyzed as described in Fig. 1. Numbers indicate the percentages of cells that have not divided, divided once, or divided twice, with the percentage calculation excluding cells that have gone through more than two divisions (see Fig. 1). Total CD4⁺Thy1.1 cells recovered from each B6 or MHCIIKO mouse were 2.9 × 10⁵ and 5.4 × 10⁵, respectively.

Figure 4

The selecting ligand does not drive CD4⁺ T cell division in irradiated hosts. 3.8 × 10⁵ CFSE-labeled CD4⁺ A^bEpTKO T cells were transferred to 450-rad sublethally irradiated hosts. Hosts were: TKO (β2M KO, wtA^bKO, IiKO); A^bEpTKO (β2M KO, wtA^bKO, IiKO, A^bEp⁺); wtA^b+DKO (β2M KO, wtA^b++, IiKO). 7 d after transfer cells were stained with PE anti-CD4 and CyC anti–TCR-β and analyzed. Histograms represent the data from live CD4⁺TCR⁺ lymph node cells.