Limits...
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

The MPECs and SLECs degranulate to a similar extent in vivo, as determined by the presence of antigens. The KLRG-1int MPECs and the KLRG-1hi SLECs were assessed for their rate of degranulation in vivo by intravenous injection of 50 µg of KLRG-1 APC and CD107a/b PE antibodies in P14 chimeric mice that had been infected with LCMV 4.75 days previously. One control set of mice were injected with IgG PE as the isotype control instead of the CD107a/b antibodies. Two hours after the antibody injection, the mice were killed, and the splenocytes were isolated and stained ex vivo with CD8, Thy1.1, the IgG isotype control or CD107a/b antibodies. (a) Representative FACS plots and summary dot plots showing the mean±s.e.m. MFI of the CD107a/b staining from two independent experiments are presented. (b) The in vivo degranulation of the MPECs and SLECs was compared at days 4.75 and 8 after LCMV infection in P14 chimeric mice. Representative FACS plots and dot plots summarizing the CD107a/b MFI are presented. Whether the difference in the mean values between the indicated groups was significant was determined using Student's unpaired t-test (*P≤0.05, **P≤0.01, ***P≤0.001). (c) The viral loads in the spleens of the mice infected with LCMV at days 4.75 and 8 are presented as bar graphs. The mean±s.e.m. are presented, and *** represents P≤0.001. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496536&req=5

fig3: The MPECs and SLECs degranulate to a similar extent in vivo, as determined by the presence of antigens. The KLRG-1int MPECs and the KLRG-1hi SLECs were assessed for their rate of degranulation in vivo by intravenous injection of 50 µg of KLRG-1 APC and CD107a/b PE antibodies in P14 chimeric mice that had been infected with LCMV 4.75 days previously. One control set of mice were injected with IgG PE as the isotype control instead of the CD107a/b antibodies. Two hours after the antibody injection, the mice were killed, and the splenocytes were isolated and stained ex vivo with CD8, Thy1.1, the IgG isotype control or CD107a/b antibodies. (a) Representative FACS plots and summary dot plots showing the mean±s.e.m. MFI of the CD107a/b staining from two independent experiments are presented. (b) The in vivo degranulation of the MPECs and SLECs was compared at days 4.75 and 8 after LCMV infection in P14 chimeric mice. Representative FACS plots and dot plots summarizing the CD107a/b MFI are presented. Whether the difference in the mean values between the indicated groups was significant was determined using Student's unpaired t-test (*P≤0.05, **P≤0.01, ***P≤0.001). (c) The viral loads in the spleens of the mice infected with LCMV at days 4.75 and 8 are presented as bar graphs. The mean±s.e.m. are presented, and *** represents P≤0.001. 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: To directly assess the in vivo cytotoxic functions of MPECs and SLECs, we employed a unique in vivo degranulation assay to measure the extent of cytotoxic granule release by the effector subsets in their native physiological environment. The assay involved direct in vivo administration of the KLRG-1 antibody to distinguish between the MPECs and SLECs, along with CD107a/b antibodies to determine the extent of degranulation (Figure 3). Optimal in vivo antibody staining with a good correlation with ex vivo staining required an incubation time of approximately 1 h for the antibodies to reach dynamic equilibrium following egress from the vasculature and accessibility of the target cells. Thus, in vivo degranulation was assessed using 1 h and 2 h incubation times. The CD107a/b staining was only seen when the antibodies were administered in vivo. Splenocytes isolated without the in vivo staining (isotype control) of CD107a/b did not exhibit positive staining when stained for CD107a/b directly ex vivo (Figure 3a). This lack of CD107a/b staining on the day 4.75 effectors ex vivo in the absence of restimulation indicated that this assay measures the degranulation of cells following in vivo antigen stimulation. Importantly, the level of CD107a/b staining following the in vivo administration of the antibodies, as assessed by MFI of the CD107a/b staining, was similar in both the MPECs and SLECs (Figure 3a). These data demonstrate that there is an equivalent antigenic encounter and elaboration of cytotoxic function by MPECs and SLECs in vivo.


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)

The MPECs and SLECs degranulate to a similar extent in vivo, as determined by the presence of antigens. The KLRG-1int MPECs and the KLRG-1hi SLECs were assessed for their rate of degranulation in vivo by intravenous injection of 50 µg of KLRG-1 APC and CD107a/b PE antibodies in P14 chimeric mice that had been infected with LCMV 4.75 days previously. One control set of mice were injected with IgG PE as the isotype control instead of the CD107a/b antibodies. Two hours after the antibody injection, the mice were killed, and the splenocytes were isolated and stained ex vivo with CD8, Thy1.1, the IgG isotype control or CD107a/b antibodies. (a) Representative FACS plots and summary dot plots showing the mean±s.e.m. MFI of the CD107a/b staining from two independent experiments are presented. (b) The in vivo degranulation of the MPECs and SLECs was compared at days 4.75 and 8 after LCMV infection in P14 chimeric mice. Representative FACS plots and dot plots summarizing the CD107a/b MFI are presented. Whether the difference in the mean values between the indicated groups was significant was determined using Student's unpaired t-test (*P≤0.05, **P≤0.01, ***P≤0.001). (c) The viral loads in the spleens of the mice infected with LCMV at days 4.75 and 8 are presented as bar graphs. The mean±s.e.m. are presented, and *** represents P≤0.001. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496536&req=5

fig3: The MPECs and SLECs degranulate to a similar extent in vivo, as determined by the presence of antigens. The KLRG-1int MPECs and the KLRG-1hi SLECs were assessed for their rate of degranulation in vivo by intravenous injection of 50 µg of KLRG-1 APC and CD107a/b PE antibodies in P14 chimeric mice that had been infected with LCMV 4.75 days previously. One control set of mice were injected with IgG PE as the isotype control instead of the CD107a/b antibodies. Two hours after the antibody injection, the mice were killed, and the splenocytes were isolated and stained ex vivo with CD8, Thy1.1, the IgG isotype control or CD107a/b antibodies. (a) Representative FACS plots and summary dot plots showing the mean±s.e.m. MFI of the CD107a/b staining from two independent experiments are presented. (b) The in vivo degranulation of the MPECs and SLECs was compared at days 4.75 and 8 after LCMV infection in P14 chimeric mice. Representative FACS plots and dot plots summarizing the CD107a/b MFI are presented. Whether the difference in the mean values between the indicated groups was significant was determined using Student's unpaired t-test (*P≤0.05, **P≤0.01, ***P≤0.001). (c) The viral loads in the spleens of the mice infected with LCMV at days 4.75 and 8 are presented as bar graphs. The mean±s.e.m. are presented, and *** represents P≤0.001. 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: To directly assess the in vivo cytotoxic functions of MPECs and SLECs, we employed a unique in vivo degranulation assay to measure the extent of cytotoxic granule release by the effector subsets in their native physiological environment. The assay involved direct in vivo administration of the KLRG-1 antibody to distinguish between the MPECs and SLECs, along with CD107a/b antibodies to determine the extent of degranulation (Figure 3). Optimal in vivo antibody staining with a good correlation with ex vivo staining required an incubation time of approximately 1 h for the antibodies to reach dynamic equilibrium following egress from the vasculature and accessibility of the target cells. Thus, in vivo degranulation was assessed using 1 h and 2 h incubation times. The CD107a/b staining was only seen when the antibodies were administered in vivo. Splenocytes isolated without the in vivo staining (isotype control) of CD107a/b did not exhibit positive staining when stained for CD107a/b directly ex vivo (Figure 3a). This lack of CD107a/b staining on the day 4.75 effectors ex vivo in the absence of restimulation indicated that this assay measures the degranulation of cells following in vivo antigen stimulation. Importantly, the level of CD107a/b staining following the in vivo administration of the antibodies, as assessed by MFI of the CD107a/b staining, was similar in both the MPECs and SLECs (Figure 3a). These data demonstrate that there is an equivalent antigenic encounter and elaboration of cytotoxic function by MPECs and SLECs in vivo.

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