Angiotensin II type-1 receptor (AT1R) regulates expansion, differentiation, and functional capacity of antigen-specific CD8+ T cells

Figures

• 

  Figure 1. AT1R is important to the expansion of antigen-specific CD8+ T cells. AT1R+/+ or AT1R−/− OT-I cells (CD8+ CD45.1+) recovered from the spleen of immunized recipient mice (CD45.2+) were analyzed on days 0, 3, 7, 12, 20, and 32 post immunization. (A) Schematics of the experimental design. 1 × 104 Naive AT1R+/+ or AT1R−/− OT-I cells (CD8+CD45.1+) were adoptively transferred to WT C57BL/6 mice (CD45.2+) recipients 1 day before intravenous inoculation with 1 × 10λ γ-irradiated P. berghei CS5M sporozoites. Mice were euthanized at the indicated time points for recovery and analysis of OT-I cells. (B) Representative CD8+CD45.1+(OT-I cells) plots gated on total lymphocytes. Percentages represent the proportion of OT-I cells (CD8+CD45.1+) among the total CD8+ T cells per spleen, recovered at days 3, 7, 12, 20, and 32 p.i. The gating strategy used for flow cytometry analysis is indicated in the Materials and Methods section. (C) Total number of AT1R+/+ (filled circle; continued line) or AT1R−/− (filled square; broken line) OT-I cells per spleen at days 3 (p = 0.136), 7 (*p = 0.044), 12 (*p = 0.003), 20 (*p = 0.0002), and 32 (p = 0.129) post inoculation, calculated as the frequencies obtained…

• 

  Figure 2. AT1R does not affect early proliferation (up to 72 h) of antigen-specific CD8+ T cells. (A) Schematics of the experimental design. WT C57BL/6 mice (CD45.2+) received 2 × 106 CFSE-labeled naive AT1R+/+ and AT1R−/− OT-I cells (CD8+CD45.1+) 1 day before inoculation with irradiated P. berghei CS5M sporozoites. (B) Mice were euthanized 3 days later, and CFSE dilutions on CD8+CD45.1+ T cells (OT-I cells) harvested from the spleen of non-imunized (naïve) and γ–spz immunized mice were analyzed by flow cytometry. Histograms represent OT-I cells taken from the spleen. (C) Number of divided OT-I cells 3 days after γ-spz inoculation, calculated by the frequency of CFSEloCD8+CD45.1+ cells in the spleen multiplied by the total number of cells obtained after spleen excision (p = 0.139). Data are means ± SEM of 4 mice per group and are representative of 3 independent experiments with similar results for each indicated time point. Abbreviation: CFSE, carboxyfluorescein diacetate succinimidyl ester.

• 

  Figure 3. AT1R upregulates IL-2 production and IL-2R expression in antigen-specific CD8+ T cells during the effector phase. 1 × 104 naive AT1R+/+ or AT1R−/− CD45.1+ OT-I cells were adoptively transferred to WT C57BL/6 (CD45.2+) recipients 1 day before IV inoculation with 1 × 105 γ-irradiated P. berghei CS5M sporozoites. IL-2R subunits (CD25 (α-chain) and CD122 (β-chain)) and IL-2 production were evaluated in AT1R+/+ and AT1R−/− OT-I cells (CD8+CD45.1+) recovered from the spleen of immunized recipient mice (CD45.2+) at indicated time points post immunization. The gating strategy used for flow cytometry analysis is indicated in the Materials and Methods section. (A,B) Representative dot plots and percentage of CD25+ OT-I cells among total OT-I cells in the spleen on days 3 (*p = 0.0289), 7 (*p = 0.0376), 12 (*p = 0.0292), 20 (p = 0.7879), and 32 (p = 0.663) post immunization. (C,D) Representative dot plots and percentage of CD122+ OT-I cells among total OT-I cells in the spleen on days 7 (*p = 0.0161), 12 (*p = 0.048), 20 (p = 0.2063), and 32 (p = 0.281) post immunization. Data are means ± SEM of 4 mice per group and are pooled from 2 independent experiments with similar results for each…

• 

  Figure 4. AT1R prevents exacerbated activation of antigen-specific CD8+ T cells during effector and memory phases. Percentage of cells expressing the markers of activation, CD69, CD160, CD44, and CD62L, were evaluated in AT1R+/+ and AT1R−/− OT-I cells (CD8+CD45.1+) recovered from the spleen of immunized recipient mice (CD45.2+) at indicated time points post immunization. The gating strategy used for flow cytometry analysis is indicated in the Materials and Methods section. (A,B) Representative dot plots and percentage of CD69+ OT-I cells among total OT-I cells in the spleen on days 3 (p = 0.4255), 7 (p = 0.9451), 12 (*p = 0.0210), 20 (*p = 0.0310), and 32 (*p = 0.0003) post immunization. (C,D) Representative dot plots and percentage of CD160+ OT-I cells among total OT-I cells in the spleen on days 7 (p = 0.9054), 12 (*p = 0.0287), 20 (*p = 0.0095), and 32 (*p = 0.0069) post immunization. Data are means ± SEM of 4 mice per group and are pooled from 2 independent experiments with similar results for each indicated day. (E,F) Representative dot plots and percentage of CD44+ OT-I cells among total OT-I cells in the spleen on days 7 (p = 0.2), 12 (p = 0.8425), 20 (*p = 0.0002), and 32 (*p =…

• 

  Figure 5. AT1R decreases exhaustion in antigen-specific CD8+ T cells. Percentage of cells expressing exhaustion markers, PD-1, LAG-3, and CTLA-4, were evaluated in AT1R+/+ and AT1R−/− OT-I cells (CD8+CD45.1+) isolated from the spleen of immunized recipient mice (CD45.2+) at indicated time points post immunization. The gating strategy used for flow cytometry analysis is indicated in the Materials and Methods section. (A,B) Representative dot plots and percentage of PD-1+ OT-I cells among total OT-I cells in the spleen on days 7 (p = 0.9), 12 (*p = 0.0192), 20 (p = 0.9), and 32 (*p = 0.0285) post immunization. (C,D) Representative dot plots and percentage of LAG-3+ OT-I cells among total OT-I cells in the spleen on days 7 (p = 0.11), 12 (*p = 0.0024), 20 (*p = 0.0468), and 32 (*p = 0.0074) post immunization. (E,F) Representative dot plots and percentage of CTLA-4+ OT-I cells among total OT-I cells in the spleen on days 7 (p = 0.6), 12 (p = 0.6907), 20 (p = 0.551), and 32 (*p = 0.0197) post immunization. Data are means ± SEM of 4 mice per group and are representative of 2 independent experiments with similar results for each indicated time point. Abbreviations: CTLA-4, cytotoxic…

• 

  Figure 6. AT1R inhibits the upregulation of IL7-Rα expression in antigen-specific CD8+ T cells during the memory development phase. Percentage of cells expressing IL-7Rα and KLRG-1 were evaluated in AT1R+/+ and AT1R−/− OT-I cells (CD8+CD45.1+) harvested from the spleen of immunized recipient mice (CD45.2+) and analyzed at the indicated time points post immunization. The gating strategy used for flow cytometry analysis is indicated in the Material and Methods section. (A,B) Representative dot plots and percentage of KLRG1+ OT-I cells among total OT-I cells in the spleen on days 7 (p = 0.5902), 12 (p = 0.07), 20 (p = 0.2654), and 32 (p = 0.8903) post immunization. (C,D) Representative dot plots and percentage of IL-7Rα + OT-I cells among total OT-I cells in the spleen on days 7 (p = 0.4388), 12 (p = 0.2857), 20 (*p = 0.0089), and 32 (*p = 0.0429) post immunization. Data are means ± SEM of 4 mice per group and are representative of 2 independent experiments with similar results for each indicated day. Abbreviations: IL7-Rα, interleukin-7 receptor α-chain; KLRG-1, killer cell lectin like receptor G1.

• 

  Figure 7. AT1R expression in effector antigen-specific CD8+ T cells fosters polyfunctional capacity. 1 × 104 naive AT1R+/+ or AT1R−/− CD45.1+ OT-I cells were adoptively transferred to WT C57BL/6 (CD45.2+) recipients 1 day before intravenous inoculation with 1 × 105 γ-irradiated P. berghei CS5M sporozoites. (A,B) Seven (A) or 12 days (B) post immunization, spleen suspensions were stimulated ex vivo with SIINFEKL peptide-coated target cells. CD8+ T-cell functionality was determined by the combinatorial analysis of the functional subsets of responding CD8+CD45.1+ T cells residing in the spleen. Responding cells are defined by the capacity to produce at least 1 of the 3 cytokines and/or surface mobilization of CD107a after ex vivo stimulation. The bar charts indicate the frequency of CD8+CD45.1+ T lymphocytes (OT-I cells) expressing each of the 15 possible combinations of CD107a, IFN-γ, TNF-α, and IL- 2 among responding OT-I cells at (A) day 7 and (B) day 12 post immunization. Bars represent means ± SD of 5 or 6 mice per group and are representative of 2 independent experiments with similar results for each indicated time point.

• 

  Figure 8. AT1R promotes cytotoxic antigen-specific CD8+ T cells during the memory phase. 1 × 104 naive AT1R+/+ or AT1R−/− CD45.1+ OT-I cells were adoptively transferred to WT C57BL/6 (CD45.2+) recipients 1 day before intravenous inoculation with 1 × 105 γ-irradiated P. berghei CS5M sporozoites. (A,B) Twenty (A) or 32 days (B) post immunization, spleen suspensions were stimulated ex vivo with SIINFEKL peptide-coated target cells. CD8+ T-cell functionality was determined by the combinatorial analysis of the functional subsets of responding CD8+CD45.1+ T cells residing in the spleen. Responding cells are defined by the capacity to produce at least 1 of the 3 cytokines and/or surface mobilization of CD107a after ex vivo stimulation. The bar charts indicate the frequency of CD8+CD45.1+ T lymphocytes (OT-I cells) expressing each of the 15 possible combinations of CD107a, IFN-γ, TNF-α, and IL- 2 among responding OT-I cells at days (A) 20 and (B) 32 post immunization. Bars represent mean ± SD of 5 or 6 mice per group and are representative of 3 independent experiments with similar results for each indicated time point.

• 

  Figure 9. AT1R induces cytotoxic memory antigen-specific CD8+ T cells in the liver. 1 × 104 naive AT1R+/+ or AT1R−/− CD45.1+ OT-I cells were adoptively transferred to WT C57BL/6 (CD45.2+) recipients 1 day before intravenous inoculation with 1 × 105 γ-irradiated P. berghei CS5M sporozoites. (A) Total number of AT1R+/+ or AT1R−/− OT-I cells per liver at days 20 (*p = 0.0073) and 32 (*p = 0.0013) post inoculation, calculated as the frequencies obtained by CD8+CD45.1+ staining, multiplied by the total number of cells obtained after liver excision. Data are means ± SEM of 4 mice per group and are representative of 2 independent experiments with similar results for each indicated time point. (B,C) Twenty (B) or 32 (C) days post immunization, T cells were recovered from the liver and were stimulated ex vivo with SIINFEKL peptide-coated target cells. CD8+ T-cell functionality was determined by the combinatorial analysis of the functional subsets of responding CD8+CD45.1+ T cells residing in the liver. Responding cells are defined by the capacity to produce at least 1 of the 3 cytokines and/or surface mobilization of CD107a after ex vivo stimulation. The bar charts indicate the frequency of…

• 

  Figure 10. Lack of AT1R expression in parasite-specific CD8+ T cells protects mice from lethal disease induced by blood-stage PbA infection. 1 × 104 naive WT or AT1R−/− CD45.1+ OT-I cells were adoptively transferred to WT C57BL/6 (CD45.2+) recipients 1 day before infection with 5 × 106 RBCs infected with OVA-PbA. (A) Schematics of the experimental design. 1 × 104 Naive WT (AT1R+/+) or AT1R−/− OT-I cells (CD8+CD45.1+) were adoptively transferred to WT C57BL/6 mice (CD45.2+) recipients 1 day before intraperitoneal injection of 5 × 106 infected red blood cells (RBCs) with P. berghei ANKA expressing ovalbumin (OVA-PbA). Mice were euthanized at the indicated time point for recovery and analysis of OT-I cells. (B) Total number of WT or AT1R−/− OT-I cells per spleen at day 6 post infection, calculated as the frequencies obtained by CD8+CD45.1+ staining, multiplied by the total number of cells obtained after spleen excision. Data are means ± SEM of 5 mice per group and are representative of 3 independent experiments with similar results (*p < 0.0001 in relation to naïve WT OT-I cells; &p < 0.0001 in relation to naïve AT1R−/− OT-I cells #p < 0.0001 in relation to WT OT-I cells). (C) CD8+ T cell…