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Antiapoptotic Role for Lifeguard in T Cell Mediated Immune Response.

Hurtado de Mendoza T, Liu F, Verma IM - PLoS ONE (2015)

Bottom Line: We observed a decreased number of LFG KO activated CD8 and CD4 T cells throughout the infection and a marked decrease in LFG KO LCMV specific memory T cells.LFG KO and WT T cells were equally sensitive to the FAS antibody Jo-2 in ex vivo cultures, and blocking extrinsic pathways of cell death in vivo with Fas L or caspase 8 inhibitors did not rescue the increased apoptosis in LFG KO T cells.Our data suggest that LFG plays a role in T cell survival during the initial phase of anti-viral immune response by protecting pre-existing memory T cells and possibly newly activated T cells resulting in a diminished immune response and a decreased number of LCMV specific memory T cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California, United States of America.

ABSTRACT
Anti-apoptotic protein Lifeguard (LFG) is upregulated on T cells upon in vitro activation. To investigate its role in T cell immunity we infected wild type and LFG knockout bone marrow chimaeras mice with LCMV. We observed a decreased number of LFG KO activated CD8 and CD4 T cells throughout the infection and a marked decrease in LFG KO LCMV specific memory T cells. WT and KO T cells proliferated at the same rate, however, LFG KO CD44(hi) T cells showed increased cell death during the initial phase of the immune response. LFG KO and WT T cells were equally sensitive to the FAS antibody Jo-2 in ex vivo cultures, and blocking extrinsic pathways of cell death in vivo with Fas L or caspase 8 inhibitors did not rescue the increased apoptosis in LFG KO T cells. Our data suggest that LFG plays a role in T cell survival during the initial phase of anti-viral immune response by protecting pre-existing memory T cells and possibly newly activated T cells resulting in a diminished immune response and a decreased number of LCMV specific memory T cells.

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Apoptosis and proliferation studies.(A) In the top panel the bar graphs represent proliferation of CD8 (left) or CD4 (right) WT and KO T cells by measuring BrdU incorporation at the pre-infection (day 0), initiation (day 3), expansion (days 6 and 8), contraction (day 15) and memory (day 35) phases of the immune response. In the bottom panel the bar graphs represent apoptotic WT and KO T cells measured by AnnexinV staining at the same timepoints described above. (B) Dot plots representing AnnexinV and DAPI staining in WT or KO CD8+ T cells at day 3 p.i. (C) The graphs in the left represent the percentage of AnnexinV+ CD8 (top) or CD4 (Bottom) T cells in WT mice divided in CD44hi (solid line, diamonds) and CD44lo (dashed line, squares) over a timecourse from day 0 to day 3 p.i. in order to monitor cell death in the initial activation phase. The right panel shows bar graphs representing the percentage of apoptotic WT and KO cells by AnnexinV staining of CD44hi CD8 (top) and CD4 (bottom) T cells at days 0 and 2 p.i. (D) Bar graphs representing the mean fluorescence intensity of LFG staining in CD44hi and lo CD8 and CD4 T cells at days 0 to 3 p.i. (n = 4 mice per timepoint, *p ≤ 0.05, **p ≤ 0.01, ± = SEM).
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pone.0142161.g003: Apoptosis and proliferation studies.(A) In the top panel the bar graphs represent proliferation of CD8 (left) or CD4 (right) WT and KO T cells by measuring BrdU incorporation at the pre-infection (day 0), initiation (day 3), expansion (days 6 and 8), contraction (day 15) and memory (day 35) phases of the immune response. In the bottom panel the bar graphs represent apoptotic WT and KO T cells measured by AnnexinV staining at the same timepoints described above. (B) Dot plots representing AnnexinV and DAPI staining in WT or KO CD8+ T cells at day 3 p.i. (C) The graphs in the left represent the percentage of AnnexinV+ CD8 (top) or CD4 (Bottom) T cells in WT mice divided in CD44hi (solid line, diamonds) and CD44lo (dashed line, squares) over a timecourse from day 0 to day 3 p.i. in order to monitor cell death in the initial activation phase. The right panel shows bar graphs representing the percentage of apoptotic WT and KO cells by AnnexinV staining of CD44hi CD8 (top) and CD4 (bottom) T cells at days 0 and 2 p.i. (D) Bar graphs representing the mean fluorescence intensity of LFG staining in CD44hi and lo CD8 and CD4 T cells at days 0 to 3 p.i. (n = 4 mice per timepoint, *p ≤ 0.05, **p ≤ 0.01, ± = SEM).

Mentions: We did not find any significant differences in BrdU positive CD8 or CD4 T cells in any of the time points analyzed (Fig 3A Top panel). This finding is supported by our ex vivo CD3 stimulated proliferation assay (S1 Fig). We labeled WT and KO splenocytes with CFSE and activated them with plate-bound CD3 and soluble CD28 for 1 to 3 days. The proliferation profile demonstrated by serial dilution of CFSE intensity was superimposable between the WT and KO cells. These finding led to our conclusion that LFG KO cells had no proliferative disadvantage. On the other hand, we found a marked increase in Annexin V positive CD8 and CD4 KO T cells at day 3 p.i. (Fig 3A Bottom panel). In the CD8 population 18.75% +/- 1% of the WT cells were apoptotic, compared to 47.42% +/- 3.79% of the KO cells (p = 0.00033) (Fig 3B). Within the CD4 T cells we saw 19.72% +/- 1.25% apoptotic cells in the WT population and 38.72% +/- 2.26% in the KO (p = 0.00032). This observation actually fits well with our initial observation that the dominance of wild type over the KO cells occurred as early as day 6 post infection, especially for CD8 cells (Fig 2A). These data suggest that LFG’s role in T cell anti-viral immune response is not through proliferation, but rather through protection from apoptosis. This protection was not afforded during the contraction phase of the immune response, where the mass majority of the apoptotic events during the immune response occur, but rather during the initial priming phase of the immune response, which none-the-less had defining quality for the rest of the immune response phases.


Antiapoptotic Role for Lifeguard in T Cell Mediated Immune Response.

Hurtado de Mendoza T, Liu F, Verma IM - PLoS ONE (2015)

Apoptosis and proliferation studies.(A) In the top panel the bar graphs represent proliferation of CD8 (left) or CD4 (right) WT and KO T cells by measuring BrdU incorporation at the pre-infection (day 0), initiation (day 3), expansion (days 6 and 8), contraction (day 15) and memory (day 35) phases of the immune response. In the bottom panel the bar graphs represent apoptotic WT and KO T cells measured by AnnexinV staining at the same timepoints described above. (B) Dot plots representing AnnexinV and DAPI staining in WT or KO CD8+ T cells at day 3 p.i. (C) The graphs in the left represent the percentage of AnnexinV+ CD8 (top) or CD4 (Bottom) T cells in WT mice divided in CD44hi (solid line, diamonds) and CD44lo (dashed line, squares) over a timecourse from day 0 to day 3 p.i. in order to monitor cell death in the initial activation phase. The right panel shows bar graphs representing the percentage of apoptotic WT and KO cells by AnnexinV staining of CD44hi CD8 (top) and CD4 (bottom) T cells at days 0 and 2 p.i. (D) Bar graphs representing the mean fluorescence intensity of LFG staining in CD44hi and lo CD8 and CD4 T cells at days 0 to 3 p.i. (n = 4 mice per timepoint, *p ≤ 0.05, **p ≤ 0.01, ± = SEM).
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Related In: Results  -  Collection

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pone.0142161.g003: Apoptosis and proliferation studies.(A) In the top panel the bar graphs represent proliferation of CD8 (left) or CD4 (right) WT and KO T cells by measuring BrdU incorporation at the pre-infection (day 0), initiation (day 3), expansion (days 6 and 8), contraction (day 15) and memory (day 35) phases of the immune response. In the bottom panel the bar graphs represent apoptotic WT and KO T cells measured by AnnexinV staining at the same timepoints described above. (B) Dot plots representing AnnexinV and DAPI staining in WT or KO CD8+ T cells at day 3 p.i. (C) The graphs in the left represent the percentage of AnnexinV+ CD8 (top) or CD4 (Bottom) T cells in WT mice divided in CD44hi (solid line, diamonds) and CD44lo (dashed line, squares) over a timecourse from day 0 to day 3 p.i. in order to monitor cell death in the initial activation phase. The right panel shows bar graphs representing the percentage of apoptotic WT and KO cells by AnnexinV staining of CD44hi CD8 (top) and CD4 (bottom) T cells at days 0 and 2 p.i. (D) Bar graphs representing the mean fluorescence intensity of LFG staining in CD44hi and lo CD8 and CD4 T cells at days 0 to 3 p.i. (n = 4 mice per timepoint, *p ≤ 0.05, **p ≤ 0.01, ± = SEM).
Mentions: We did not find any significant differences in BrdU positive CD8 or CD4 T cells in any of the time points analyzed (Fig 3A Top panel). This finding is supported by our ex vivo CD3 stimulated proliferation assay (S1 Fig). We labeled WT and KO splenocytes with CFSE and activated them with plate-bound CD3 and soluble CD28 for 1 to 3 days. The proliferation profile demonstrated by serial dilution of CFSE intensity was superimposable between the WT and KO cells. These finding led to our conclusion that LFG KO cells had no proliferative disadvantage. On the other hand, we found a marked increase in Annexin V positive CD8 and CD4 KO T cells at day 3 p.i. (Fig 3A Bottom panel). In the CD8 population 18.75% +/- 1% of the WT cells were apoptotic, compared to 47.42% +/- 3.79% of the KO cells (p = 0.00033) (Fig 3B). Within the CD4 T cells we saw 19.72% +/- 1.25% apoptotic cells in the WT population and 38.72% +/- 2.26% in the KO (p = 0.00032). This observation actually fits well with our initial observation that the dominance of wild type over the KO cells occurred as early as day 6 post infection, especially for CD8 cells (Fig 2A). These data suggest that LFG’s role in T cell anti-viral immune response is not through proliferation, but rather through protection from apoptosis. This protection was not afforded during the contraction phase of the immune response, where the mass majority of the apoptotic events during the immune response occur, but rather during the initial priming phase of the immune response, which none-the-less had defining quality for the rest of the immune response phases.

Bottom Line: We observed a decreased number of LFG KO activated CD8 and CD4 T cells throughout the infection and a marked decrease in LFG KO LCMV specific memory T cells.LFG KO and WT T cells were equally sensitive to the FAS antibody Jo-2 in ex vivo cultures, and blocking extrinsic pathways of cell death in vivo with Fas L or caspase 8 inhibitors did not rescue the increased apoptosis in LFG KO T cells.Our data suggest that LFG plays a role in T cell survival during the initial phase of anti-viral immune response by protecting pre-existing memory T cells and possibly newly activated T cells resulting in a diminished immune response and a decreased number of LCMV specific memory T cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California, United States of America.

ABSTRACT
Anti-apoptotic protein Lifeguard (LFG) is upregulated on T cells upon in vitro activation. To investigate its role in T cell immunity we infected wild type and LFG knockout bone marrow chimaeras mice with LCMV. We observed a decreased number of LFG KO activated CD8 and CD4 T cells throughout the infection and a marked decrease in LFG KO LCMV specific memory T cells. WT and KO T cells proliferated at the same rate, however, LFG KO CD44(hi) T cells showed increased cell death during the initial phase of the immune response. LFG KO and WT T cells were equally sensitive to the FAS antibody Jo-2 in ex vivo cultures, and blocking extrinsic pathways of cell death in vivo with Fas L or caspase 8 inhibitors did not rescue the increased apoptosis in LFG KO T cells. Our data suggest that LFG plays a role in T cell survival during the initial phase of anti-viral immune response by protecting pre-existing memory T cells and possibly newly activated T cells resulting in a diminished immune response and a decreased number of LCMV specific memory T cells.

Show MeSH
Related in: MedlinePlus