Figure 2. Requirement for CD4 cell interaction with host class II for CD8 tolerance.

BM-reconstituted mice prepared as in Fig. 1 received allogeneic B10.A BMT. (A) The incidence of chimerism in the B cell lineage at various time points. Chimerism was defined as ≥5% donor cells among B220⁺ B cells. One representative experiment out of three is shown (7-8 animals/group/experiment). (B) Chimerism levels (±SEM) over time for B cells and CD4 T cells in PBL. (C) Mice treated as in panel A also received depleting anti-CD8 on Day -1. CD8 T cells were <0.5% of PBL by 2 weeks post-BMT. The incidence of B cell chimerism is shown at indicated times. One representative experiment of two is shown (6-7 animals/group/experiment). (D) Chimerism levels (±SEM) over time are shown for blood B cells and CD4 T cells for groups in panel C. (E) Representative FACS plots for measuring chimerism are shown 6 weeks post-BMT for a chimeric WT mouse (upper row) and a non-chimeric mouse lacking MHC class II on recipient APCs (lower row) from the experiment shown in panel B. Donor cells are marked with FITC-labelled anti-D^d mAb 34-2-12. The panels showing CD4 and CD8 T cells and B220+ B cells are gated on lymphocytes; the panels showing CD11b+ cells are gated on granulocytes. Percentages indicate the relative frequencies in the total lymphocyte or granulocyte population, respectively. Chimerism was then calculated as the percentage of donor MHC-positive cells versus total cells in a certain lineage (e.g. for CD4 cells: [14.8/(14.8 + 11.5)] * 100 = 56.3%).