S. Strikingly, the transition to TA 02 site memory resulted in no functional avidity maturation. Instead, the low functional avidity of bim2/2 SMARTAs was maintained at memory time points (Fig. 3D), showing that merely enabling the survival of CD4+ effector Th1 populations into the memory compartment does not ensure the acquisition of memory function. Thus, following infection with a particular pathogen, Bim can promote CD4+ T cell survival during the transition to memory, but the development of memory KDM5A-IN-1 site function is Bim-independent, as evidenced by the survival of Bim-deficient SMARTA memory cells that were profoundly dysfunctional.bim2/2 SMARTA “Memory” Cells Lack the Ability to Respond to Secondary ChallengeTo directly test their memory function, we rechallenged Lmgp61-generated bim2/2 SMARTA memory cells either homologously with Lm-gp61 or heterologously with LCMV or Vac-GP. Whether rechallenged with Lm-gp61, Vac-GP or LCMV, bim2/2 SMARTA memory cells failed to significantly expand as compared to the endogenous memory cells in the same host (Fig. 4A). Similarly, at day 5 post-rechallenge, bim2/2 SMARTA memory cells demonstrated consistently poor effector function, as measured by their ability to make multiple cytokines upon restimulation (IFNc, TNFa and IL-2). bim2/2 SMARTA secondary responders continued to be largely comprised of 16985061 IFNc monoproducers, in sharp contrast to the multiple cytokine production of polyclonal endogenous secondary responders (Fig. 4B and C). This dysfunctional phenotype was maintained throughout the course of the recall response (data not shown).DiscussionOverall, our findings demonstrate that Bim itself is capable of intrinsically mediating the death of functionally defective, low avidity SMARTA effector Th1 cells generated following Lm-gp61 infection. bim2/2 SMARTA cells were able to survive beyond the effector phase and maintain themselves similarly to endogenous responders in the same host, yet they failed to acquire theBim Shapes the Functional CD4+ Memory PoolFigure 2. Bim mediates the elimination of SMARTA cells following Lm-gp61 infection. We co-transferred 56103 each WT SMARTA (Thy1.1+ Thy1.2+) and bim2/2 SMARTA (Thy1.1+) into B6 hosts (Thy1.2+), followed by infection with either Lm-gp61 or Vac-GP one day later. A and C, Representative plots indicate expansion and survival of SMARTA cells in the spleen following Lm-gp61 or Vac-GP infection. B and D, Graph indicates the survival of WT or bim2/2 SMARTA cells in the spleen following Lm-gp61 infection. Dashed line indicates the limit of detection. Results are representative of 3? mice per group per time point and four independent experiments. E, Mixed bone marrow chimeras, generated using a 1:1 mix of wildtype (CD45.1+) and Bin-deficient (Thy1.1+) bone marrow injected into lethally irradiated B6 (Thy1.2+CD45.2+) hosts, were infected with Lmgp61 8?0 weeks post-transplant. The number of IFNc-producing Th1 effector or memory cells in the spleen was determined at 7 or 42 days posttransplant. F, Splenocytes harvested at the indicated time points were stimulated with decreasing concentration of GP61?0 peptide for four hours ex vivo in the presence of Brefeldin A, followed by intracellular antibody staining for IFNc. Bar graphs indicate the effective peptide concentration required to elicit the half maximal response. Error bars indicate the SEM (n = 4 mice/group at each time point). p values for statistically significant differences were calculated by a two-tailed Student’.S. Strikingly, the transition to memory resulted in no functional avidity maturation. Instead, the low functional avidity of bim2/2 SMARTAs was maintained at memory time points (Fig. 3D), showing that merely enabling the survival of CD4+ effector Th1 populations into the memory compartment does not ensure the acquisition of memory function. Thus, following infection with a particular pathogen, Bim can promote CD4+ T cell survival during the transition to memory, but the development of memory function is Bim-independent, as evidenced by the survival of Bim-deficient SMARTA memory cells that were profoundly dysfunctional.bim2/2 SMARTA “Memory” Cells Lack the Ability to Respond to Secondary ChallengeTo directly test their memory function, we rechallenged Lmgp61-generated bim2/2 SMARTA memory cells either homologously with Lm-gp61 or heterologously with LCMV or Vac-GP. Whether rechallenged with Lm-gp61, Vac-GP or LCMV, bim2/2 SMARTA memory cells failed to significantly expand as compared to the endogenous memory cells in the same host (Fig. 4A). Similarly, at day 5 post-rechallenge, bim2/2 SMARTA memory cells demonstrated consistently poor effector function, as measured by their ability to make multiple cytokines upon restimulation (IFNc, TNFa and IL-2). bim2/2 SMARTA secondary responders continued to be largely comprised of 16985061 IFNc monoproducers, in sharp contrast to the multiple cytokine production of polyclonal endogenous secondary responders (Fig. 4B and C). This dysfunctional phenotype was maintained throughout the course of the recall response (data not shown).DiscussionOverall, our findings demonstrate that Bim itself is capable of intrinsically mediating the death of functionally defective, low avidity SMARTA effector Th1 cells generated following Lm-gp61 infection. bim2/2 SMARTA cells were able to survive beyond the effector phase and maintain themselves similarly to endogenous responders in the same host, yet they failed to acquire theBim Shapes the Functional CD4+ Memory PoolFigure 2. Bim mediates the elimination of SMARTA cells following Lm-gp61 infection. We co-transferred 56103 each WT SMARTA (Thy1.1+ Thy1.2+) and bim2/2 SMARTA (Thy1.1+) into B6 hosts (Thy1.2+), followed by infection with either Lm-gp61 or Vac-GP one day later. A and C, Representative plots indicate expansion and survival of SMARTA cells in the spleen following Lm-gp61 or Vac-GP infection. B and D, Graph indicates the survival of WT or bim2/2 SMARTA cells in the spleen following Lm-gp61 infection. Dashed line indicates the limit of detection. Results are representative of 3? mice per group per time point and four independent experiments. E, Mixed bone marrow chimeras, generated using a 1:1 mix of wildtype (CD45.1+) and Bin-deficient (Thy1.1+) bone marrow injected into lethally irradiated B6 (Thy1.2+CD45.2+) hosts, were infected with Lmgp61 8?0 weeks post-transplant. The number of IFNc-producing Th1 effector or memory cells in the spleen was determined at 7 or 42 days posttransplant. F, Splenocytes harvested at the indicated time points were stimulated with decreasing concentration of GP61?0 peptide for four hours ex vivo in the presence of Brefeldin A, followed by intracellular antibody staining for IFNc. Bar graphs indicate the effective peptide concentration required to elicit the half maximal response. Error bars indicate the SEM (n = 4 mice/group at each time point). p values for statistically significant differences were calculated by a two-tailed Student’.
