Fig. 1. Characterization of polyclonal alloreactive CD8 memory T cell subsets.

Thy1.1 mice were immunized with BALB/c splenocytes (3 × 10⁷, i.p.) and spleen and LN cells were harvested 6 – 12 weeks later to sort for CD8 T_CM and T_EM. (A) Sorting of CD8 memory T cell subsets. CD8 memory T cell subsets were sorted after gating on CD8⁺CD44^highCD62L^high cells (CD8 T_CM) and CD8⁺CD44^highCD62L^low cells (CD8 T_EM). Sorted cells were tested for purity prior to adoptive transfer. (B) Phenotype of CD8 T_CM and T_EM. Expression of CD69, 1B11, CD25, CD27, CD127 and CD122 on CD8 T_CM and T_EM is shown (representative FACS plots of 5 – 6 experiments). (C) Ex-vivo IFNγ production by CD8 T_CM and T_EM. Sorted CD8 T_CM and T_EM were assessed for IFNγ production by intracellular cytokine staining after 5-hr ex-vivo stimulation with BALB/c splenocytes. IFNγ production within the Thy1.1⁺ population is shown after gating on CD8⁺ T cells. Naïve T cells from unimmunized Thy1.1 mice (Naïve) and unfractionated sorted CD8⁺ CD44^high memory T cells (CD8 memory) from immunized Thy1.1 mice that contained both CD62L^high and CD62L^low cells were used as controls. Representative FACS plots of 4 – 5 experiments are shown. (D) Quantitation of alloreactive IFNγ⁺ T cells within CD8 T_CM and T_EM subsets after 5-hr ex-vivo stimulation with BALB/c splenocytes. Percent of IFNγ⁺ T cells within naïve, unfractionated CD8 memory (memory) and sorted CD8 T_CM and T_EM populations. Mean ± SD of 4 – 5 experiments. (E) Alloreactive IFNγ⁺ T cells within CD8 T_CM and T_EM subsets upon ex-vivo stimulation with BALB/c splenocytes over time. Percent of IFNγ⁺ T cells within naïve, sorted CD8 T_CM and T_EM populations after ex-vivo stimulation with BALB/c splenocytes at 6hrs, 12hrs and 24hrs. (F) In-vivo cytotoxicity of alloreactive CD8 T_CM and T_EM subsets in wt adoptive hosts. Wt mice were treated with anti-NK1.1 (PK136, 300µg, i.p.) to deplete NK cells. 3-days later, sorted CD8 T_CM (1.9 × 10⁶) or CD8 T_EM (5 × 10⁵) containing similar numbers of BALB/c-reactive IFNγ⁺ T cells were transferred to wt adoptive hosts followed by injection of equal numbers of CFSE labeled H-2^b (5 × 10⁶, 2µM B6, syngeneic) and H-2^d (5 × 10⁶, 0.2µM, BALB/c, allogeneic) splenocytes on the same day. 12-hrs later, in vivo killing of target cells was measured in harvested spleen and LN cells as loss of H-2^d target cells compared to loss of H-2^b syngeneic cells in adoptive hosts of memory T cells (T_CM or T_EM) versus naïve B6 control mice (as described in Materials and Methods) (representative FACS plots of n = 3 mice/grp are shown). (G) Quantitation of in-vivo specific lysis of allogeneic cells by alloreactive CD8 T_CM and T_EM subsets in wt adoptive hosts. Percent of BALB/c-cell lysis in harvested spleen and LNs from wt adoptive hosts of CD8 T_CM or T_EM. Mean ± SD of n = 3 mice/grp. (H) Expression of chemokine and adhesion receptors on CD8 T_CM and T_EM. Expression of CCR7, CXCR3, CCR5, CD49d (α₄-integrin), β₁-integrin, α₄β₇ and CD11a (LFA-1) on CD8 T_CM and T_EM subsets shown in comparison to CD8⁺ CD44^low naïve T cells (representative FACS plots of 3 – 4 experiments).