T cell receptor-dependent regulation of lipid rafts controls naive CD8+ T cell homeostasis - PubMed (original) (raw)

T cell receptor-dependent regulation of lipid rafts controls naive CD8+ T cell homeostasis

Jae-Ho Cho et al. Immunity. 2010.

Abstract

T cell receptor (TCR) contact with self ligands keeps T cells alive and is shown here to cause naive CD8(+), but not CD4(+), T cells to be hypersensitive to certain gamma(c) cytokines, notably interleukin (IL)-2, IL-15, and IL-7. Hypersensitivity of CD8(+) T cells to IL-2 was dependent on a low-level TCR signal, associated with high expression of CD5 and GM1, a marker for lipid rafts, and was abolished by disruption of lipid rafts. By contrast, CD4(+) T cells expressed low amounts of GM1 and were unresponsive to IL-2. Physiologically, sensitivity to IL-7 and IL-15 maintains survival of resting CD8(+) T cells, whereas sensitivity to IL-2 may be irrelevant for normal homeostasis but crucial for the immune response. Thus, TCR contact with antigen upregulates GM1 and amplifies responsiveness of naive CD8(+) T cells to IL-2, thereby making the cells highly sensitive to exogenous IL-2 from CD4(+) T helper cells.

Copyright 2010 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Proliferation and differentiation of naive CD8+ cells exposed to cytokines in vitro. (A and B) CFSE-labeled (A) or unlabeled (B) purified naive (CD44lo) B6 CD4+ or CD8+ T cells were cultured with the indicated cytokines (all 1 μg/ml except IFNβ used at 2 × 104 units/ml) and analyzed on day 5 for proliferation by flow cytometry (A) and [3H]thymidine incorporation (B). (C and D) Proliferation kinetics (C) and viable cell counts (D) for purified B6 naive CD8+ cells cultured with cross-linked anti-CD3 mAb (5 μg/ml), IL-2 (1 μg/ml) or IL-15 (1 μg/ml) by [3H]thymidine uptake (C) and trypan blue exclusion (D). (E) Proliferation of naive B6, 2C or OT-I CD8+ cells on day 5 after culture with or without 1 μg/ml IL-2 or IL-15 (F) Expression of activation markers on B6 naive CD8+ cells stimulated with cross-linked anti-CD3 mAb (5 μg/ml) or IL-2 (1 μg/ml). (G) Intracellular cytokine production and granzyme B expression were analyzed for naive B6 CD8+ cells cultured for 3 days with the indicated stimuli (1st stimuli) as described in Supplemental Experimental Procedures. Data (A–G) are representative of at least three independent experiments (B–E; mean and SD of triplicate samples).

Figure 2

Figure 2

Response of naive CD8+ cells to IL-2 depends on TCR/self-MHC-I interaction. (A and B) Proliferation (A) and CFSE dilution and granzyme B expression (B) of naive B6, HY (from HY.Rag−/− mice) or P14 CD8+ cells on day 6 after culture with various (0.05–1.5 μg/ml; A) or fixed (1.5 μg/ml; B) concentrations of IL-2. (C) Proliferation of purified MHC-I+/+ (from normal B6) or MHC-I−/− (from MHC-I−/−→B6 BM chimeras) naive CD8+ cells on days 3 and 6 after stimulation with IL-2. (D and E) CFSE dilution and granzyme B expression of purified MHC-I+/+ (Thy1.1; from B6) and MHC-I−/− (Thy1.2; from BM chimeras in C) naive CD8+ cells either cultured separately (D) or cocultured (E) for 5 days with IL-2. (F) Proliferation of naive CD8+ cells from B6 (MHC-I+/+), BM chimeras in C (MHC-I−/−) or MHC-I−/− mice injected with MHC-I−/− CD8+ cells (from BM chimeras in C) 3 days before (MHC-I−/− → MHC-I−/−) was analyzed on day 4 after culture with IL-2. (G) Proliferation of B6 naive CD8+ cells (Ly5.1) parked for 3 days and recovered from B6 (B6 → B6), IL-7−/− (B6 → IL-7−/−) or TAP-1−/− (B6 → TAP-1−/−) mice on day 4 after culture with IL-2 or on day 3 with cross-linked anti-CD3 mAb. Data (A–G) are representative of 2–3 experiments (C, F and G; mean ± SD of triplicate samples).

Figure 3

Figure 3

Levels of CD5 on naive CD8+ cells correlate with the strength of responsiveness to cytokines in vitro and in vivo. (A–D) Purified naive CD5lo and CD5hi CD8+ cells from B6 (A–D), 2C (A and D) or HY (from HY.Rag+/+ mice; A and D) mice were cultured with cross-linked anti-CD3 mAb (for 3 days; A) or IL-2 (for days 3 and 5, B; for day 5, C; and for day 6, D) and analyzed for proliferation (A, B and D) or for expression of activation markers (C). (E) CFSE-labeled purified B6 naive CD5lo (Ly5.1) and CD5hi (Thy1.1) CD8+ cells were cotransferred into irradiated B6 or RAG−/− mice and 6 days later, pooled SP and LN were analyzed for CFSE dilution (left), percentage of donor cells that underwent >3 rounds of division, and total donor cell recovery (middle and right, respectively; mean and SD of three mice per group). (F) Proliferation of CFSE-labeled B6 naive CD5lo and CD5hi CD8+ cells on day 7 after culture with IL-7 (50 ng/ml), IL-12 (50 ng/ml) or both. Data (A–F) are representative of 2–3 experiments (A, B and D; mean ± SD of triplicate samples).

Figure 4

Figure 4

GM1 expression on T cell subsets, and the effects of disrupting lipid rafts on the ability of naive CD8+ cells to respond to IL-2. (A and B) Proliferation (A) and % inhibition of proliferation (B) of MβCD-treated purified naive B6 CD8+ cells were analyzed on day 2 after culture with the indicated stimuli as described in Supplemental Experimental Procedures. Data show the mean ± SD of triplicate samples. (C) GM1 levels (MFI) on CD44hi CD8+, CD44lo CD5lo vs. CD44lo CD5hi CD8+ cells from HY.Rag+/+ mice analyzed by flow cytometry. Data show the mean and SD of five mice. (D) GM1 MFI levels on CD44lo vs. CD44hi subsets (left) or naive (CD44lo) CD5lo vs. CD5hi subsets (right) of B6 CD4+ and CD8+ cells. Each circle represents an individual mouse and the line indicates the mean. (E) Proliferation of purified B6 naive GM1lo and GM1hi CD8+ cells on day 3 after culture with IL-2 or on day 1 with cross-linked anti-CD3 mAb. Data show the mean ± SD of triplicate samples. (F) CFSE-labeled purified B6 naive GM1lo (Ly5.1) and GM1hi (Thy1.1) CD8+ cells were cotransferred into irradiated B6 mice and 7 days later, pooled SP and LN were analyzed for CFSE dilution (left), % of donor cells that underwent >4 rounds of division, and total donor cell recovery (middle and right, respectively; mean and SD of three mice). Data (A–F) are representative of 2–3 independent experiments.

Figure 5

Figure 5

Expression of GM1 and CD5 on T cell subsets during ontogeny. (A–C) GM1 (A and B) and CD5 (C) MFI levels on B6 DP, SP CD4+ and SP CD8+ thymocytes (A and C) or on CD24lo vs. CD24hi subsets of SP CD4+ and CD8+ thymocytes (B). (D) GM1 (left) and CD5 (right) MFI levels on CD44lo CD4+ vs. CD44lo CD8+ cells in B6 LN. Data (A–D; mean and SD of three to five mice) are representative of 3 experiments. (E) GM1 MFI levels of B6 naive CD4+ and CD8+ cells (Ly5.1) cotransferred into normal B6 or TAP-1−/− mice 1 or 3 days before; pooled SP and LN were analyzed by flow cytometry. Data (mean ± SD of 2–3 mice per group at each time point) are representative of 3 experiments.

Figure 6

Figure 6

Culturing naive CD8+ cells with IL-2 induces lipid raft (GM1) clustering and colocalization of GM1 with IL-2Rβ. (A) B6 naive CD8+ cells were untreated or treated for 15 min with the indicated stimuli and analyzed for lipid raft clustering by GM1 confocal staining with FITC-conjugated CTB (green). (B) GM1 confocal staining of CD5lo vs. CD5hi subsets of B6 naive CD4+ and CD8+ cells untreated or treated for 15 min with IL-2 (1 μg/ml). (C) GM1 confocal staining of B6 naive CD8+ cells cultured for 15 min with or without the indicated γc cytokines (1 μg/ml). (D) B6 naive CD8+ cells were cultured for 15 min with IL-2 (1 μg/ml) and analyzed for colocalization (yellow when merged) of GM1 lipid rafts (green) and IL-2Rβ (red). Data (A–D) are representative of 2–3 experiments.

Figure 7

Figure 7

Hypersensitivity of CD8+ cells to IL-2 augments their capacity to respond to foreign antigens. (A) Proliferation of B6 naive CD5lo and CD5hi CD8+ cells to a surrogate weak antigen; cells were cultured for 2 days with or without soluble anti-CD3 mAb (0.1 μg/ml) with graded concentrations of IL-2 (0.3–10 ng/ml). Data show the mean ± SD of triplicate samples. (B) Proliferation of CFSE-labeled 2C naive CD5lo (Ly5.1) vs. CD5hi (Thy1.1) CD8+ cells to a strong antigen; cells were cultured with allogeneic BALB/c splenocytes with or without IL-2 blockade and analyzed by flow cytometry. (C) Proliferation of CFSE-labeled 2C naive CD5lo (Ly5.1) vs. CD5hi (Thy1.1) CD8+ cells to a weak antigen; cells as in B were cocultured with CFSE-labeled OT-II naive CD4+ cells and syngeneic B6 splenocytes pulsed with 20 μM p2Ca peptide with or without OVA323-339 peptide (OT-IIp). (D) Surface markers on T cells after TCR stimulation were examined by culturing CD44lo CD8+ and CD4+ cells for 20 hr in plates coated with graded concentrations of anti-CD3 mAb and then stained for the markers shown. Data (A–D) are representative of 2–4 experiments.

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