S T test. **p#0.01, *p#0.05. doi:10.1371/journal.pone.0067363.gfunctional characteristics of a memory population. bim2/2 SMARTA cells demonstrated and maintained poor effector function 10781694 when restimulated with peptide and failed to mount substantial in vivo recall responses MedChemExpress Benzocaine following rechallenge. Thus, while Bim is required to regulate the survival of poorly functional SMARTA cells following Lm-gp61 infection, it alone is not sufficient to restore their ability to become fully functional memory cells. One caveat to the use of SMARTA transgenic T cells is the possibility that they are not representative of polyclonal endogenous Th1 effector and memory cells. Our studies of endogenous Bim-deficient CD4+ T cells, however, similarly suggest that theabsence of Homatropine methobromide contraction by Bim-deficient T cells corresponds to the rescue and entry of memory cells into the memory pool with poor functional avidity. Overall, our results highlight a key function for Bim in functionally shaping the Th1 memory repertoire. While Bim has been found to have a role in mediating activated T cell contraction after antigen clearance following infection with certain pathogens, the signals that lead to Bim-mediated apoptosis in most CD4+ T cells but not those fated to enter the memory pool remain unknown. Our prior findings indicated that Bim expression was clonally selective, depending on the infectious model. In those prior studies, the differential ability of LCMV- or Lm-gp61-Bim Shapes the Functional CD4+ Memory PoolFigure 3. Persisting bim2/2 SMARTA “memory” cells are functionally defective. We analyzed the functionality of SMARTA responses in the spleen following Lm-gp61 infection. A, Representative plots indicate the expression of IFNc and TNFa by WT or bim2/2 SMARTA cells in the spleen at the indicated time points after infection with Lm-gp61. B, Bars graph indicate the shift in MFI of IFNc-producing cells, as compared to unstimulated controls. C, Bar graph indicates the percent of IFNc-producing SMARTA cells that also make TNFa and IL-2 (“triple producers”). D, Graphs display the frequency of IFNc-producing SMARTA cells or polyclonal endogeneous CD4+ T cells specific for the same epitope over a range of peptide concentrations as a percentage of the maximal response (defined as the response at the highest peptide concentration). Results are representative of 3? mice per group per time point and four independent experiments. Error bars indicate the SEM. doi:10.1371/journal.pone.0067363.ginduced SMARTA effector Th1 cells to survive into the memory pool corresponded strongly (and inversely) with the expression of Bim transcripts [14]. Here we show a required mechanistic role for Bim in the elimination of dysfunctional SMARTA Th1 cells induced by Lm-gp61. Because these are monoclonal populations, one possibility is that Bim activity, and subsequent Bim-regulatedsurvival, are influenced by the qualitative or quantitative nature of the TCR-mediated activation signal during primary activation. Little is known about how the nature or timing TCR signals may influence the decision of a CD4+ T cell to enter a Bimmediated cell death pathway. Previous work from our lab has shown that by as early as day five post Lm-gp61 infection,Bim Shapes the Functional CD4+ Memory PoolFigure 4. bim2/2 SMARTA “Memory” cells lack the ability to respond to secondary challenge. Lm-gp61 immune mice (day 90 postinfection) containing “memory” bim2/2 SMARTA were rechallenged with LCMV, Vac-GP.S T test. **p#0.01, *p#0.05. doi:10.1371/journal.pone.0067363.gfunctional characteristics of a memory population. bim2/2 SMARTA cells demonstrated and maintained poor effector function 10781694 when restimulated with peptide and failed to mount substantial in vivo recall responses following rechallenge. Thus, while Bim is required to regulate the survival of poorly functional SMARTA cells following Lm-gp61 infection, it alone is not sufficient to restore their ability to become fully functional memory cells. One caveat to the use of SMARTA transgenic T cells is the possibility that they are not representative of polyclonal endogenous Th1 effector and memory cells. Our studies of endogenous Bim-deficient CD4+ T cells, however, similarly suggest that theabsence of contraction by Bim-deficient T cells corresponds to the rescue and entry of memory cells into the memory pool with poor functional avidity. Overall, our results highlight a key function for Bim in functionally shaping the Th1 memory repertoire. While Bim has been found to have a role in mediating activated T cell contraction after antigen clearance following infection with certain pathogens, the signals that lead to Bim-mediated apoptosis in most CD4+ T cells but not those fated to enter the memory pool remain unknown. Our prior findings indicated that Bim expression was clonally selective, depending on the infectious model. In those prior studies, the differential ability of LCMV- or Lm-gp61-Bim Shapes the Functional CD4+ Memory PoolFigure 3. Persisting bim2/2 SMARTA “memory” cells are functionally defective. We analyzed the functionality of SMARTA responses in the spleen following Lm-gp61 infection. A, Representative plots indicate the expression of IFNc and TNFa by WT or bim2/2 SMARTA cells in the spleen at the indicated time points after infection with Lm-gp61. B, Bars graph indicate the shift in MFI of IFNc-producing cells, as compared to unstimulated controls. C, Bar graph indicates the percent of IFNc-producing SMARTA cells that also make TNFa and IL-2 (“triple producers”). D, Graphs display the frequency of IFNc-producing SMARTA cells or polyclonal endogeneous CD4+ T cells specific for the same epitope over a range of peptide concentrations as a percentage of the maximal response (defined as the response at the highest peptide concentration). Results are representative of 3? mice per group per time point and four independent experiments. Error bars indicate the SEM. doi:10.1371/journal.pone.0067363.ginduced SMARTA effector Th1 cells to survive into the memory pool corresponded strongly (and inversely) with the expression of Bim transcripts [14]. Here we show a required mechanistic role for Bim in the elimination of dysfunctional SMARTA Th1 cells induced by Lm-gp61. Because these are monoclonal populations, one possibility is that Bim activity, and subsequent Bim-regulatedsurvival, are influenced by the qualitative or quantitative nature of the TCR-mediated activation signal during primary activation. Little is known about how the nature or timing TCR signals may influence the decision of a CD4+ T cell to enter a Bimmediated cell death pathway. Previous work from our lab has shown that by as early as day five post Lm-gp61 infection,Bim Shapes the Functional CD4+ Memory PoolFigure 4. bim2/2 SMARTA “Memory” cells lack the ability to respond to secondary challenge. Lm-gp61 immune mice (day 90 postinfection) containing “memory” bim2/2 SMARTA were rechallenged with LCMV, Vac-GP.