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Effector memory Th1 CD4 T cells are maintained in a mouse model of chronic malaria.

Stephens R, Langhorne J - PLoS Pathog. (2010)

Bottom Line: CD4(+) memory T cells (CD44(hi)IL-7Rα(+)) developed during the chronic infection, and were readily distinguishable from effector (CD62L(lo)IL-7Rα(-)) cells in acute infection.On the basis of cell surface phenotype, we classified memory CD4(+) T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62L(lo)CD27(+)) dominated the chronic infection.We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, London, UK.

ABSTRACT
Protection against malaria often decays in the absence of infection, suggesting that protective immunological memory depends on stimulation. Here we have used CD4(+) T cells from a transgenic mouse carrying a T cell receptor specific for a malaria protein, Merozoite Surface Protein-1, to investigate memory in a Plasmodium chabaudi infection. CD4(+) memory T cells (CD44(hi)IL-7Rα(+)) developed during the chronic infection, and were readily distinguishable from effector (CD62L(lo)IL-7Rα(-)) cells in acute infection. On the basis of cell surface phenotype, we classified memory CD4(+) T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62L(lo)CD27(+)) dominated the chronic infection. We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells. In adoptive transfer, CD44(hi) memory cells from chronically infected mice were more effective at delaying and reducing parasitemia and pathology than memory cells from drug-treated mice without chronic infection, and contained a greater proportion of effector cells producing IFN-γ and TNFα, which may have contributed to the enhanced protection. These findings may explain the observation that in humans with chronic malaria, activated effector memory cells are best maintained in conditions of repeated exposure.

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Related in: MedlinePlus

Memory T cells with an activated phenotype can be distinguished from effector T cells.Sorted Naïve (CD44loCD25−), CFSE-labelled B5 T cells (2×106) were seeded into congenic Thy1.1+ mice. Mice were infected with 105 P. chabaudi iRBC, sacrificed and splenocytes stained with anti-CD4, Thy1.2 and activation markers CD44, CD43, CD27, IL7Rα and CD62L at different times post infection. A) Divided B5 T cells (CFSEneg Thy1.2+, left plot and histogram) were analyzed for expression of the activation markers on day 9 post-infection by multiparameter flow cytometry. Histograms show gating for the activation markers. CFSEneg B5 Tg effector T cells (Teff) are CD44int-hi, IL-7Rα−, CD62Llo, and can be CD43+/−, and CD27+/−. B) CFSEneg (divided) B5 Tg T cells were boolean gated into 32 possible subsets based on their expression all five markers. The percentage of cells expressing each combination of markers for all of the subsets at day 9 (white bars) and day 60 (black bars) are shown in the histograms. Boxes below indicate the marker listed to the left. The blue box on the right indicates the group with none of the markers of activation. C) CFSEneg Tg cells were pooled into groups according to the number of activation markers expressed (color coded for 0–5 markers of activation as shown in the panel on the left, with colors ranging from blue: no activation markers; to purple, 5 activation markers), and the percentage of cells in each group is shown in a pie chart to indicate the relative activation state on a given day post-infection. A representative experiment of 3 performed is shown. The values shown are for 2–5 mice at each time point, cytometry data were concatenated. Experiment was repeated twice with similar results.
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ppat-1001208-g001: Memory T cells with an activated phenotype can be distinguished from effector T cells.Sorted Naïve (CD44loCD25−), CFSE-labelled B5 T cells (2×106) were seeded into congenic Thy1.1+ mice. Mice were infected with 105 P. chabaudi iRBC, sacrificed and splenocytes stained with anti-CD4, Thy1.2 and activation markers CD44, CD43, CD27, IL7Rα and CD62L at different times post infection. A) Divided B5 T cells (CFSEneg Thy1.2+, left plot and histogram) were analyzed for expression of the activation markers on day 9 post-infection by multiparameter flow cytometry. Histograms show gating for the activation markers. CFSEneg B5 Tg effector T cells (Teff) are CD44int-hi, IL-7Rα−, CD62Llo, and can be CD43+/−, and CD27+/−. B) CFSEneg (divided) B5 Tg T cells were boolean gated into 32 possible subsets based on their expression all five markers. The percentage of cells expressing each combination of markers for all of the subsets at day 9 (white bars) and day 60 (black bars) are shown in the histograms. Boxes below indicate the marker listed to the left. The blue box on the right indicates the group with none of the markers of activation. C) CFSEneg Tg cells were pooled into groups according to the number of activation markers expressed (color coded for 0–5 markers of activation as shown in the panel on the left, with colors ranging from blue: no activation markers; to purple, 5 activation markers), and the percentage of cells in each group is shown in a pie chart to indicate the relative activation state on a given day post-infection. A representative experiment of 3 performed is shown. The values shown are for 2–5 mice at each time point, cytometry data were concatenated. Experiment was repeated twice with similar results.

Mentions: The acute P. chabaudi infection lasting about 20 days, and the subsequent low-level chronic infection may result in the continuous generation of effector cells, as has been reported in other persistent infections [9], [21], [26]. Therefore, we utilized multiparameter flow cytometry and the activation and memory markers, CD44, CD43, CD27, IL-7Rα and CD62L [16], [27], [28] to distinguish between CD4+ effector and memory subsets. At day 9 of infection, the majority of Tg cells had divided (CFSEneg, FACS gating showing level of CD44 expression correlating with cell division is shown in Figure S1), and were defined as effector cells (Teff) CD62Llo, IL-7Rα−, CD43+, and CD27−, and CD44int (Figure 1A). Flow cytometry data gated on CD4+Thy1.2+ CFSEneg divided B5 Tg cells were collected at several time-points during the infection, and subjected to boolean gating analysis, which distributed them into the 32 possible subsets generated by testing every possible combination of the 5 activation markers. This is shown in the histogram in Figure 1B, where the percentage of the divided B5 Tg CD4+ T cells falling into each subset is illustrated. Strikingly, this unbiased analysis identified effector cells as CD62LloIL-7Rα− during the acute parasitemia at day 9, while memory cells were easily distinguished by their upregulation of IL-7Rα, as seen in the majority of divided, CFSEneg B5 Tg cells by day 60, which fell into both the effector (Tem) and central (Tcm) memory categories as indicated by their expression of CD62L. Various subsets could be seen within these major populations, and the other three markers, CD44, CD43, CD27, were used to determine the overall activation status of the cells. By grouping subsets based on the number of activation markers they express, we constructed pie charts to demonstrate graphically how activated the MSP1-specific cells were throughout the P. chabaudi infection (Figure 1C). More than 80% of the Tg T cells expressed markers of activation at day 9, and interestingly, many malaria-specific cells remained activated even at day 21, when the parasite was cleared to below 0.01% of erythrocytes infected. At day 45, the activation status of the cells appeared to have stabilized, so that even at day 60, 25% of the cells maintained three or more markers of activation. This suggests that a large proportion of the MSP1-specific Tg T cells that persisted late into the chronic phase of infection remained considerably activated and did not become resting or central memory cells in this time.


Effector memory Th1 CD4 T cells are maintained in a mouse model of chronic malaria.

Stephens R, Langhorne J - PLoS Pathog. (2010)

Memory T cells with an activated phenotype can be distinguished from effector T cells.Sorted Naïve (CD44loCD25−), CFSE-labelled B5 T cells (2×106) were seeded into congenic Thy1.1+ mice. Mice were infected with 105 P. chabaudi iRBC, sacrificed and splenocytes stained with anti-CD4, Thy1.2 and activation markers CD44, CD43, CD27, IL7Rα and CD62L at different times post infection. A) Divided B5 T cells (CFSEneg Thy1.2+, left plot and histogram) were analyzed for expression of the activation markers on day 9 post-infection by multiparameter flow cytometry. Histograms show gating for the activation markers. CFSEneg B5 Tg effector T cells (Teff) are CD44int-hi, IL-7Rα−, CD62Llo, and can be CD43+/−, and CD27+/−. B) CFSEneg (divided) B5 Tg T cells were boolean gated into 32 possible subsets based on their expression all five markers. The percentage of cells expressing each combination of markers for all of the subsets at day 9 (white bars) and day 60 (black bars) are shown in the histograms. Boxes below indicate the marker listed to the left. The blue box on the right indicates the group with none of the markers of activation. C) CFSEneg Tg cells were pooled into groups according to the number of activation markers expressed (color coded for 0–5 markers of activation as shown in the panel on the left, with colors ranging from blue: no activation markers; to purple, 5 activation markers), and the percentage of cells in each group is shown in a pie chart to indicate the relative activation state on a given day post-infection. A representative experiment of 3 performed is shown. The values shown are for 2–5 mice at each time point, cytometry data were concatenated. Experiment was repeated twice with similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991260&req=5

ppat-1001208-g001: Memory T cells with an activated phenotype can be distinguished from effector T cells.Sorted Naïve (CD44loCD25−), CFSE-labelled B5 T cells (2×106) were seeded into congenic Thy1.1+ mice. Mice were infected with 105 P. chabaudi iRBC, sacrificed and splenocytes stained with anti-CD4, Thy1.2 and activation markers CD44, CD43, CD27, IL7Rα and CD62L at different times post infection. A) Divided B5 T cells (CFSEneg Thy1.2+, left plot and histogram) were analyzed for expression of the activation markers on day 9 post-infection by multiparameter flow cytometry. Histograms show gating for the activation markers. CFSEneg B5 Tg effector T cells (Teff) are CD44int-hi, IL-7Rα−, CD62Llo, and can be CD43+/−, and CD27+/−. B) CFSEneg (divided) B5 Tg T cells were boolean gated into 32 possible subsets based on their expression all five markers. The percentage of cells expressing each combination of markers for all of the subsets at day 9 (white bars) and day 60 (black bars) are shown in the histograms. Boxes below indicate the marker listed to the left. The blue box on the right indicates the group with none of the markers of activation. C) CFSEneg Tg cells were pooled into groups according to the number of activation markers expressed (color coded for 0–5 markers of activation as shown in the panel on the left, with colors ranging from blue: no activation markers; to purple, 5 activation markers), and the percentage of cells in each group is shown in a pie chart to indicate the relative activation state on a given day post-infection. A representative experiment of 3 performed is shown. The values shown are for 2–5 mice at each time point, cytometry data were concatenated. Experiment was repeated twice with similar results.
Mentions: The acute P. chabaudi infection lasting about 20 days, and the subsequent low-level chronic infection may result in the continuous generation of effector cells, as has been reported in other persistent infections [9], [21], [26]. Therefore, we utilized multiparameter flow cytometry and the activation and memory markers, CD44, CD43, CD27, IL-7Rα and CD62L [16], [27], [28] to distinguish between CD4+ effector and memory subsets. At day 9 of infection, the majority of Tg cells had divided (CFSEneg, FACS gating showing level of CD44 expression correlating with cell division is shown in Figure S1), and were defined as effector cells (Teff) CD62Llo, IL-7Rα−, CD43+, and CD27−, and CD44int (Figure 1A). Flow cytometry data gated on CD4+Thy1.2+ CFSEneg divided B5 Tg cells were collected at several time-points during the infection, and subjected to boolean gating analysis, which distributed them into the 32 possible subsets generated by testing every possible combination of the 5 activation markers. This is shown in the histogram in Figure 1B, where the percentage of the divided B5 Tg CD4+ T cells falling into each subset is illustrated. Strikingly, this unbiased analysis identified effector cells as CD62LloIL-7Rα− during the acute parasitemia at day 9, while memory cells were easily distinguished by their upregulation of IL-7Rα, as seen in the majority of divided, CFSEneg B5 Tg cells by day 60, which fell into both the effector (Tem) and central (Tcm) memory categories as indicated by their expression of CD62L. Various subsets could be seen within these major populations, and the other three markers, CD44, CD43, CD27, were used to determine the overall activation status of the cells. By grouping subsets based on the number of activation markers they express, we constructed pie charts to demonstrate graphically how activated the MSP1-specific cells were throughout the P. chabaudi infection (Figure 1C). More than 80% of the Tg T cells expressed markers of activation at day 9, and interestingly, many malaria-specific cells remained activated even at day 21, when the parasite was cleared to below 0.01% of erythrocytes infected. At day 45, the activation status of the cells appeared to have stabilized, so that even at day 60, 25% of the cells maintained three or more markers of activation. This suggests that a large proportion of the MSP1-specific Tg T cells that persisted late into the chronic phase of infection remained considerably activated and did not become resting or central memory cells in this time.

Bottom Line: CD4(+) memory T cells (CD44(hi)IL-7Rα(+)) developed during the chronic infection, and were readily distinguishable from effector (CD62L(lo)IL-7Rα(-)) cells in acute infection.On the basis of cell surface phenotype, we classified memory CD4(+) T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62L(lo)CD27(+)) dominated the chronic infection.We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, London, UK.

ABSTRACT
Protection against malaria often decays in the absence of infection, suggesting that protective immunological memory depends on stimulation. Here we have used CD4(+) T cells from a transgenic mouse carrying a T cell receptor specific for a malaria protein, Merozoite Surface Protein-1, to investigate memory in a Plasmodium chabaudi infection. CD4(+) memory T cells (CD44(hi)IL-7Rα(+)) developed during the chronic infection, and were readily distinguishable from effector (CD62L(lo)IL-7Rα(-)) cells in acute infection. On the basis of cell surface phenotype, we classified memory CD4(+) T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62L(lo)CD27(+)) dominated the chronic infection. We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells. In adoptive transfer, CD44(hi) memory cells from chronically infected mice were more effective at delaying and reducing parasitemia and pathology than memory cells from drug-treated mice without chronic infection, and contained a greater proportion of effector cells producing IFN-γ and TNFα, which may have contributed to the enhanced protection. These findings may explain the observation that in humans with chronic malaria, activated effector memory cells are best maintained in conditions of repeated exposure.

Show MeSH
Related in: MedlinePlus