Figure 1. Development of alloreactive memory T cells is impaired in B-cell deficient mice.

A. Rejection of BALB/c (H-2^d) skin allografts transplanted to µMT (H-2^b) and wt (H-2^b) mice (MST = 16 and 15 days, respectively; p > 0.05; n = 10 mice/grp). Quantitiation of alloreactive effector (B) and memory (D) T cells in µMT and wt mice. Spleen (SP), lymph node (LN) and bone marrow (BM) cells were harvested from BALB/c skin grafted µMT and wt recipients at 14-days (effector phase) and at 8-weeks after rejection (memory phase) for quantitation of alloreactive T cells. Harvested cells were re-stimulated ex-vivo for 6-hrs with BALB/c splenocytes and IFNγ producing CD4 and CD8 T cells were assessed by flow cytometry and enumerated (Mean ± SD; *, p < 0.05; n = 4 mice/grp). C. CD8 memory precursors within alloreactive effector T cells in µMT and wt mice. Representative FACS plots of IL-7Rα expression on BALB/c-reactive IFNγ⁺ population within CD8⁺ CD44^hi splenic T cells harvested at 14-days after BALB/c skin transplantation are shown. E–F. Cytotoxic function of µMT and wt memory T cells was assessed by in-vivo (E) and in-vitro (F) allogeneic cell lysis at 8-weeks after BALB/c skin graft rejection (memory phase). µMT and wt mice were depleted of NK cells and equal numbers of CFSE labeled H-2^b (2 × 10⁷, 2µM B6 wt, syngeneic) and H-2^d (2 × 10⁷, 0.2µM, BALB/c, allogeneic) splenocytes were injected (i.v.). 24-hrs later, in-vivo killing of BALB/c cells in comparison to B6 cells was measured by flow cytometry (Mean ± SD; **, p < 0.005; n = 3 – 4 mice/grp). Purified T cells from SP and LN cells of µMT and wt memory mice were incubated with calcein labeled BALB/c splenocytes (0.3mM, 100:1) and 4-hrs later, in-vitro killing of BALB/c cells was measured by flow cytometry (Mean ± SD; *, p < 0.05; n = 3 –4 mice/grp). G. Allograft rejection in memory µMT and wt mice. At 8-weeks after BALB/c allograft rejection (memory phase), µMT and wt recipients were re-challenged with BALB/c skin grafts and treated with DST (2 × 10⁷) and anti-CD40L (MR1, 1mg on days 0, 7 and 14 after transplantation). Allograft rejection was assessed in µMT and wt memory mice, and compared to naïve mice (MST = 30, 20 and 30 days, respectively; p < 0.05; n = 4 – 9 mice/grp).