Figure 1.
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.
Figure 2.
Effector CD4+ T cells persist in P. chabaudi infection and early effector memory cells predominate.
Naïve (CD44loCD25−), CFSE-labelled B5 T cells (2×106) were seeded into congenic Thy1.1+ mice, which were then infected with 105 P. chabaudi iRBC. A) Divided B5 cells (CFSEneg) were analyzed for expression of CD62L and IL-7Rα to measure the percentages of Teff (CD62Llo, IL-7Rα−) in the spleen on day 9 (middle panel) and day 60 (right panel). As an internal negative control, naïve cells (CFSE+) from uninfected mice are shown (left panel). B) CD44hiIL-7Rα+ memory cells are shown in the divided CFSEneg population. C) Kinetics of appearance of Teff and Tmem as defined in A) and B) by percentage (left panel) or number (×103, right panel). D) Tmem were subdivided using CD62L and CD27 to measure central (Tcm, CD62LhiCD27+), and early effector memory cells (TemE, CD62LloCD27+) as well as CD27− late effector memory T cells (TemL, CD62LloCD27−) shown here at day 60 of infection. The relative proportions of Tcm, TemE and TemL of infection are shown in the right graph. E) PD-1 expression on B5 Transgenic CD4+ T cells seeded in Thy1.1 congenic mice at different times during P. chabaudi infection. All PD-1+ cells were CD62Llo. Graphs represent the means and SEM of 2 to 5 mice per time point. Plots show CD4+Thy1.2+CFSEneg cells concatenated from 2–5 mice per time point (gated as shown in Figure 1A, raw data for B, D shown in Figure S3). Experiments were repeated three times with similar results.
Figure 3.
Heterogeneity of cytokine production and differentiation potential of the memory CD4+ T-cell subsets in chronic P. chabaudi infection.
A) and B) Naïve (CD44loCD25−), CFSE-labelled B5 T cells (2×106) were seeded into congenic Thy1.1+ mice to follow the antigen-specific cytokine response in memory cell subsets. Mice were infected with 105 P. chabaudi infected RBC and CD4+ T cells analyzed 60 days later. Divided B5 cells (CD44hi CFSEneg) were analyzed for A) expression of CD27 and CD62L to identify memory T cell subsets (Tcm, CD62LhiCD27+), (Temearly (TemE), CD62LloCD27+) and (Temlate (TemL), CD62LloCD27−) and production of IL-10 and IFN-γ in each subset. B) Divided B5 T cells (CFSEneg) were analyzed for CD44 and CD62L to identify Tem (CD44hiCD62Llo) and Tcm (CD44hiCD62Lhi) and production of IL-2 and TNF-α. These data are representative of 5 individual mice. C) and D) Purified memory T cell subsets (CD4+CD44hiIL-7Rα+); Tcm, (CD62LhiCD27+), (TemE, CD62LloCD27+) and (TemL, CD62LloCD27+) were transferred (105) into RAG° mice which were then infected with P. chabaudi. Spleens were analysed on day 38 after infection by multi-parameter FACS analysis; C) memory cell composition on recovery, Tcm, TemE and TemL, as defined in A), of CD4+ T cells, shown as percentage of lymphocytes; D) Proposed linear differentiation pathway for memory T cell subsets based on the data in C).
Figure 4.
Chronically stimulated memory T cells protect immunodeficient mice from parasitemia and pathology better than rested memory T cells.
MSP1-specific CD4+ B5 memory (CD44hi) or naïve (CD44lowCD25−) T cells were purified from B5 TCR Tg mice infected 2.5 months previously and harbouring a chronic infection (−CQ, closed symbol/bar), or drug treated from days 30–34 to remove the chronic infection (+CQ, open symbol/bar). The cells (105) were transferred into RAG° recipients together with immune B cells (107) and the recipients infected with 5×104 P. chabaudi iRBC. A) Parasitemia (geometric mean +/−SEM) is at onset of patent parasitemia (day 6, left graph), and at peak parasitemia (days 8 and 10, middle graph) B) Peak weight change is shown as a measure of cachexia (left graph), maximum loss of body temperature (middle graph) and P.chabaudi -specific IgG antibodies (day 21) measured in the serum of the transferred RAG° animals by ELISA. C) Serum IL-10, IFN-γ, and TNFα were measured on day 7 in RAG° mice receiving Tg cells from chronically infected (closed symbols) or drug-treated mice (open symbols). The data shown are the means and SEM of 4–7 mice.
Figure 5.
Treatment of a chronic P. chabaudi infection changes the phenotype of memory cells and their cytokine profiles towards Th1 late effector profile.
CD4+ MSP1-specific B5 Tg T cells were seeded into Thy1.1 congenic mice to follow the antigen-specific response. Mice were infected with 105 Plasmodium chabaudi and treated with Chloroquine (CQ) during days 30–34 to clear the chronic infection. Splenocytes were analyzed by flow cytometry on day 60 post-infection, and the FACS plots were gated on Thy1.2+CD4+ cells. A) Differences in the proportions of Tmem, (CD44hi IL-7Rα+), and effector (Teff, CD44hi IL-7Rα−) cells at day 60 between mice treated and not treated with chloroquine (+CQ and −CQ respectively). The percentage of IL-7Rα+ Tmem or IL-7Rα− Teff within the CD44hi cell population is shown as a graph (right). These differences were observed in 4 independent experiments. B) IFN-γ and TNFα production in Thy1.2+CD4+ CD44hiCD62Llo B5 cells from chloroquine-treated and -untreated mice at day 60 post infection. C) Multiple cytokine producing B5 CD4 T cells from chloroquine-treated and untreated mice at day 60 post-infection. Percent double-producing TNFα+IFN-γ+IL-2− and triple-producing IFN-γ+TNFα+IL-2+ are shown. D) IFNγ+ TNFα+ (IL-2−) double producing cells within the Tem subset of B5 at 60 days post-infection. The graphs show means and SEM from 5 mice. ** p≤.01 and * p≤.05 by student's t-test.