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Early CD8 T-cell memory precursors and terminal effectors exhibit equipotent in vivo degranulation.

Yuzefpolskiy Y, Baumann FM, Kalia V, Sarkar S - Cell. Mol. Immunol. (2014)

Bottom Line: Early after priming, effector CD8 T cells are distinguished into memory precursor and short-lived effector cell subsets (MPECs and SLECs).Here, we delineated a distinct in vivo heterogeneity in killer cell lectin-like receptor G1 (KLRG-1) expression, which was strongly associated with diverse MPEC and SLEC fates.These data provide direct in vivo evidence that memory-fated cells pass through a robust effector phase.

View Article: PubMed Central - PubMed

ABSTRACT
Early after priming, effector CD8 T cells are distinguished into memory precursor and short-lived effector cell subsets (MPECs and SLECs). Here, we delineated a distinct in vivo heterogeneity in killer cell lectin-like receptor G1 (KLRG-1) expression, which was strongly associated with diverse MPEC and SLEC fates. These in vivo MPECs and SLECs expressed equivalent levels of cytotoxic molecules and effector cytokines. Using a unique in vivo degranulation assay, we found that the MPECs and SLECs similarly encountered infected target cells and elaborated equivalent levels of cytotoxicity in vivo. These data provide direct in vivo evidence that memory-fated cells pass through a robust effector phase. Additionally, the preferential localization of the MPECs in the lymph nodes, where a lesser degree of cytotoxicity was elaborated, suggests that the MPECs may be protected from excessive stimulation and terminal differentiation by virtue of their differential tissue localization. These data provide novel mechanistic insights into the linear decreasing potential model of memory differentiation.

No MeSH data available.


Related in: MedlinePlus

In vivo stained KLRG-1 heterogeneous effectors represent the MPEC and SLEC populations that express similar levels of effector molecules. At day 4.75 after the LCMV infection, splenocytes were isolated from the P14 chimeric mice that had been stained for KLRG-1 PE in vivo as described in Figure 1. The CD8+ T cells were sorted into KLRG-1int and KLRG-1hi subsets, and approximately 1×106 donor cells were adoptively transferred into congenically mismatched C57Bl/6 recipient mice that had been infected with LCMV 4.75 days earlier (infection-matched recipients). (a) Antigen-specific donor CD8 T cells were analyzed in the PBMCs of the recipient mice at the indicated days after the adoptive transfer and are presented as line graphs. The average±s.e.m. values are plotted from two independent repeats. Unpaired Student's t-tests were used for the analysis of statistical significance, and P≤0.001 is depicted by ***. (b) The KLRG-1int and KLRG-1hi donor cells were enumerated in the indicated tissues at approximately day 50 after the adoptive transfer by a flow cytometry analysis of the CD8+Thy1.1+ T cells. The bar graphs show the average number of donor cells at memory±s.e.m. Unpaired Student's t-tests were used for the analysis of statistically significant differences between the KLRG-1int and KLRG-1hi groups (*P≤0.05, **P≤0.01, ***P≤0.001). The in vivo stained effector CD8 T cells from the day 4.75 LCMV-infected P14 chimeric mice were FACS purified into KLRG-1int and KLRG-1hi subsets. (c) The level of granzyme B expression in both subsets was assessed by ex vivo intracellular staining and a flow cytometry analysis. Histogram plots with the MFI of granzyme B expression are shown. (d) The purified effector subsets were also stimulated with 0.2 µg/ml of GP33-41 peptide for 5 h in the presence of brefeldin A, followed by intracellular cytokine staining for IFN-γ and TNF-α. The MFIs of the indicated cytokines that were stained in both effector subsets are presented as bar graphs. FACS, fluorescence-activated cell sorting; KLRG-1, killer cell lectin-like receptor G1; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; MPEC, memory precursor effector cell; PBMC, peripheral blood mononuclear cell; SLEC, short-lived effector cell.
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fig2: In vivo stained KLRG-1 heterogeneous effectors represent the MPEC and SLEC populations that express similar levels of effector molecules. At day 4.75 after the LCMV infection, splenocytes were isolated from the P14 chimeric mice that had been stained for KLRG-1 PE in vivo as described in Figure 1. The CD8+ T cells were sorted into KLRG-1int and KLRG-1hi subsets, and approximately 1×106 donor cells were adoptively transferred into congenically mismatched C57Bl/6 recipient mice that had been infected with LCMV 4.75 days earlier (infection-matched recipients). (a) Antigen-specific donor CD8 T cells were analyzed in the PBMCs of the recipient mice at the indicated days after the adoptive transfer and are presented as line graphs. The average±s.e.m. values are plotted from two independent repeats. Unpaired Student's t-tests were used for the analysis of statistical significance, and P≤0.001 is depicted by ***. (b) The KLRG-1int and KLRG-1hi donor cells were enumerated in the indicated tissues at approximately day 50 after the adoptive transfer by a flow cytometry analysis of the CD8+Thy1.1+ T cells. The bar graphs show the average number of donor cells at memory±s.e.m. Unpaired Student's t-tests were used for the analysis of statistically significant differences between the KLRG-1int and KLRG-1hi groups (*P≤0.05, **P≤0.01, ***P≤0.001). The in vivo stained effector CD8 T cells from the day 4.75 LCMV-infected P14 chimeric mice were FACS purified into KLRG-1int and KLRG-1hi subsets. (c) The level of granzyme B expression in both subsets was assessed by ex vivo intracellular staining and a flow cytometry analysis. Histogram plots with the MFI of granzyme B expression are shown. (d) The purified effector subsets were also stimulated with 0.2 µg/ml of GP33-41 peptide for 5 h in the presence of brefeldin A, followed by intracellular cytokine staining for IFN-γ and TNF-α. The MFIs of the indicated cytokines that were stained in both effector subsets are presented as bar graphs. FACS, fluorescence-activated cell sorting; KLRG-1, killer cell lectin-like receptor G1; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; MPEC, memory precursor effector cell; PBMC, peripheral blood mononuclear cell; SLEC, short-lived effector cell.

Mentions: A higher KLRG-1 expression level in the direct ex vivo stains has typically been associated with more terminally differentiated cells, such that the differential expression of KLRG-1 marks the diverse memory and death fates of the effector CD8 T cells.6,9 We next assessed the fates associated with the in vivo stained KLRG-1int and KLRG-1hi effector cells. The in vivo stained effector CD8 T cells were FACS purified at day 4.5 after infection and were adoptively transferred into infection-matched recipient mice. Infection-matched congenically distinct mice were employed as recipients to ensure that the donor cells differentiated in similarly infected environments from where they were purified. The donor cells were followed longitudinally in the blood, and the final numbers were determined at memory. As shown in Figure 2a, both effector subsets continued to expand to similar extents after transfer and increased their numbers by approximately 10-fold until the peak of the effector responses. However, following antigen clearance, the KLRG-1int subset underwent a lesser degree of contraction and preferentially survived into the memory phase compared to the KLRG-1hi effectors. Following contraction, the final numbers of KLRG-1int donor cells were also significantly higher in all lymphoid and non-lymphoid tissues analyzed at memory (Figure 2b). At the peak of the donor cell expansion, neither subset of the unstained donor cells had any of the remaining original KLRG-1 antibody stain that was used for sorting (data not shown). Thus, the KLRG-1int and KLRG-1hi donors underwent contraction and began to exhibit survival differences >5 days after the adoptive transfer when no detectable cell surface bound antibodies remained, suggesting that the differences in cell survivability were likely independent of the differential levels of cell surface bound KLRG-1 antibodies used during FACS purification. Consistent with ex vivo studies,6,9 these data clearly demonstrate that in vivo effector cell heterogeneity in KLRG-1 expression marks distinct MPEC and SLEC fates.


Early CD8 T-cell memory precursors and terminal effectors exhibit equipotent in vivo degranulation.

Yuzefpolskiy Y, Baumann FM, Kalia V, Sarkar S - Cell. Mol. Immunol. (2014)

In vivo stained KLRG-1 heterogeneous effectors represent the MPEC and SLEC populations that express similar levels of effector molecules. At day 4.75 after the LCMV infection, splenocytes were isolated from the P14 chimeric mice that had been stained for KLRG-1 PE in vivo as described in Figure 1. The CD8+ T cells were sorted into KLRG-1int and KLRG-1hi subsets, and approximately 1×106 donor cells were adoptively transferred into congenically mismatched C57Bl/6 recipient mice that had been infected with LCMV 4.75 days earlier (infection-matched recipients). (a) Antigen-specific donor CD8 T cells were analyzed in the PBMCs of the recipient mice at the indicated days after the adoptive transfer and are presented as line graphs. The average±s.e.m. values are plotted from two independent repeats. Unpaired Student's t-tests were used for the analysis of statistical significance, and P≤0.001 is depicted by ***. (b) The KLRG-1int and KLRG-1hi donor cells were enumerated in the indicated tissues at approximately day 50 after the adoptive transfer by a flow cytometry analysis of the CD8+Thy1.1+ T cells. The bar graphs show the average number of donor cells at memory±s.e.m. Unpaired Student's t-tests were used for the analysis of statistically significant differences between the KLRG-1int and KLRG-1hi groups (*P≤0.05, **P≤0.01, ***P≤0.001). The in vivo stained effector CD8 T cells from the day 4.75 LCMV-infected P14 chimeric mice were FACS purified into KLRG-1int and KLRG-1hi subsets. (c) The level of granzyme B expression in both subsets was assessed by ex vivo intracellular staining and a flow cytometry analysis. Histogram plots with the MFI of granzyme B expression are shown. (d) The purified effector subsets were also stimulated with 0.2 µg/ml of GP33-41 peptide for 5 h in the presence of brefeldin A, followed by intracellular cytokine staining for IFN-γ and TNF-α. The MFIs of the indicated cytokines that were stained in both effector subsets are presented as bar graphs. FACS, fluorescence-activated cell sorting; KLRG-1, killer cell lectin-like receptor G1; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; MPEC, memory precursor effector cell; PBMC, peripheral blood mononuclear cell; SLEC, short-lived effector cell.
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fig2: In vivo stained KLRG-1 heterogeneous effectors represent the MPEC and SLEC populations that express similar levels of effector molecules. At day 4.75 after the LCMV infection, splenocytes were isolated from the P14 chimeric mice that had been stained for KLRG-1 PE in vivo as described in Figure 1. The CD8+ T cells were sorted into KLRG-1int and KLRG-1hi subsets, and approximately 1×106 donor cells were adoptively transferred into congenically mismatched C57Bl/6 recipient mice that had been infected with LCMV 4.75 days earlier (infection-matched recipients). (a) Antigen-specific donor CD8 T cells were analyzed in the PBMCs of the recipient mice at the indicated days after the adoptive transfer and are presented as line graphs. The average±s.e.m. values are plotted from two independent repeats. Unpaired Student's t-tests were used for the analysis of statistical significance, and P≤0.001 is depicted by ***. (b) The KLRG-1int and KLRG-1hi donor cells were enumerated in the indicated tissues at approximately day 50 after the adoptive transfer by a flow cytometry analysis of the CD8+Thy1.1+ T cells. The bar graphs show the average number of donor cells at memory±s.e.m. Unpaired Student's t-tests were used for the analysis of statistically significant differences between the KLRG-1int and KLRG-1hi groups (*P≤0.05, **P≤0.01, ***P≤0.001). The in vivo stained effector CD8 T cells from the day 4.75 LCMV-infected P14 chimeric mice were FACS purified into KLRG-1int and KLRG-1hi subsets. (c) The level of granzyme B expression in both subsets was assessed by ex vivo intracellular staining and a flow cytometry analysis. Histogram plots with the MFI of granzyme B expression are shown. (d) The purified effector subsets were also stimulated with 0.2 µg/ml of GP33-41 peptide for 5 h in the presence of brefeldin A, followed by intracellular cytokine staining for IFN-γ and TNF-α. The MFIs of the indicated cytokines that were stained in both effector subsets are presented as bar graphs. FACS, fluorescence-activated cell sorting; KLRG-1, killer cell lectin-like receptor G1; LCMV, lymphocytic choriomeningitis virus; MFI, mean fluorescence intensity; MPEC, memory precursor effector cell; PBMC, peripheral blood mononuclear cell; SLEC, short-lived effector cell.
Mentions: A higher KLRG-1 expression level in the direct ex vivo stains has typically been associated with more terminally differentiated cells, such that the differential expression of KLRG-1 marks the diverse memory and death fates of the effector CD8 T cells.6,9 We next assessed the fates associated with the in vivo stained KLRG-1int and KLRG-1hi effector cells. The in vivo stained effector CD8 T cells were FACS purified at day 4.5 after infection and were adoptively transferred into infection-matched recipient mice. Infection-matched congenically distinct mice were employed as recipients to ensure that the donor cells differentiated in similarly infected environments from where they were purified. The donor cells were followed longitudinally in the blood, and the final numbers were determined at memory. As shown in Figure 2a, both effector subsets continued to expand to similar extents after transfer and increased their numbers by approximately 10-fold until the peak of the effector responses. However, following antigen clearance, the KLRG-1int subset underwent a lesser degree of contraction and preferentially survived into the memory phase compared to the KLRG-1hi effectors. Following contraction, the final numbers of KLRG-1int donor cells were also significantly higher in all lymphoid and non-lymphoid tissues analyzed at memory (Figure 2b). At the peak of the donor cell expansion, neither subset of the unstained donor cells had any of the remaining original KLRG-1 antibody stain that was used for sorting (data not shown). Thus, the KLRG-1int and KLRG-1hi donors underwent contraction and began to exhibit survival differences >5 days after the adoptive transfer when no detectable cell surface bound antibodies remained, suggesting that the differences in cell survivability were likely independent of the differential levels of cell surface bound KLRG-1 antibodies used during FACS purification. Consistent with ex vivo studies,6,9 these data clearly demonstrate that in vivo effector cell heterogeneity in KLRG-1 expression marks distinct MPEC and SLEC fates.

Bottom Line: Early after priming, effector CD8 T cells are distinguished into memory precursor and short-lived effector cell subsets (MPECs and SLECs).Here, we delineated a distinct in vivo heterogeneity in killer cell lectin-like receptor G1 (KLRG-1) expression, which was strongly associated with diverse MPEC and SLEC fates.These data provide direct in vivo evidence that memory-fated cells pass through a robust effector phase.

View Article: PubMed Central - PubMed

ABSTRACT
Early after priming, effector CD8 T cells are distinguished into memory precursor and short-lived effector cell subsets (MPECs and SLECs). Here, we delineated a distinct in vivo heterogeneity in killer cell lectin-like receptor G1 (KLRG-1) expression, which was strongly associated with diverse MPEC and SLEC fates. These in vivo MPECs and SLECs expressed equivalent levels of cytotoxic molecules and effector cytokines. Using a unique in vivo degranulation assay, we found that the MPECs and SLECs similarly encountered infected target cells and elaborated equivalent levels of cytotoxicity in vivo. These data provide direct in vivo evidence that memory-fated cells pass through a robust effector phase. Additionally, the preferential localization of the MPECs in the lymph nodes, where a lesser degree of cytotoxicity was elaborated, suggests that the MPECs may be protected from excessive stimulation and terminal differentiation by virtue of their differential tissue localization. These data provide novel mechanistic insights into the linear decreasing potential model of memory differentiation.

No MeSH data available.


Related in: MedlinePlus