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T Cell Receptor-Dependent Regulation of Lipid Rafts Controls Naive CD8+ T Cell Homeostasis Immunity 32(2) 26 February 2010 Jae-Ho Cho, Hee-Ok Kim, Charles D. Surh and Jonathan Sprent Lee, Hye-Yeong Introduction • Naive T cells are kept alive through continuous T cell receptor (TCR) interaction with major histocompatibility complex (MHC) molecules complexed with various self peptides • Such TCR-MHC interaction plus contact with interleukin (IL)-7 causes lowlevel signaling, which promotes long-term survival of T cells in interphase through synthesis of antiapoptotic molecules such as Bcl-2 • lymphopenia-induced ‘‘homeostatic’’ proliferation (LIP) reflects a rise in amounts of IL-7 and serves to replenish the T cell pool size • IL-7-driven LIP in lymphopenic hosts is characteristically slow • Recently, a rapid form of homeostatic proliferation has been observed following T cell transfer to mice lacking components of the IL-2 receptor (IL-2R) Introduction • The physiological relevance of naive T cell responsiveness to IL-2 and IL15 and why such responsiveness is MHC dependent is unknown • To assess this issue, we have studied stimulation of naive T cells with cytokines in vitro and examined the role of monosialotetrahexosylganglioside (GM1)-containing lipid rafts. Introduction Lipid Rafts • plasma membrane of cells made of a combination of glycosphingolipids and protein receptors organized in glycolipoprotein microdomains Figure 1. Proliferation and Differentiation of Naive CD8+ T Cells Exposed to Cytokines In Vitro [3H] incorporation Figure 1. Proliferation and Differentiation of Naive CD8+ T Cells Exposed to Cytokines In Vitro [3H] incorporation trypan blue day 5 cultured with > upregulation of activation markers • IL-2-stimulated CD8+ T cells showed strong effector function in terms of both cytokine (IFN-g and TNF-a) and granzyme B synthesis • This finding was surprising because the cells were not subjected to TCR ligation. Figure 2. Response of Naive CD8+ T Cells to IL-2 Depends on TCR-Self-MHC-I Interaction > IL-2-induced proliferation of naïve CD8 T cells in vivo require TCR interaction with self-MHC-I (Cho et al., 2007) >> purified CD8 T cell의 IL-2에 대한 반응은 T cell 간의 interaction에 의존적일 것이다. > 각 mouse(HY.Rag2-/-/P14/B6)에서 얻은 naïve CD8 T cells(CD44lo, CD8+) > culture with IL-2 Figure 2. Response of Naive CD8+ T Cells to IL-2 Depends on TCR-Self-MHC-I Interaction WT/KO 따로 WT와 KO을 함께 Tap1-/ MHC-I lo Figure 3. Levels of CD5 on Naive CD8+ T Cells Correlate with the Strength of Responsiveness to Cytokines in Vitro and In Vivo • CD8 T cell responses to IL-2 in vitro required continuous TCR-MHC-I interaction T cell-deficient hosts에서 poor homeostatic proliferation을 보이는 TCR Tg CD8+ T cells은 CD5의 expression level이 낮게 나타난다.(Kieper et al., 2004) >> IL-2에 다르게 반응하는 normal B6 CD8 T cell의 차이가 바로 CD5의 level 차이가 아닐까 • Figure 3. Levels of CD5 on Naive CD8+ T Cells Correlate with the Strength of Responsiveness to Cytokines in Vitro and In Vivo >> CD3 engagement에는 차이 없음 IL-2Rb >> activation marker 차이 >> IL-2에 대해서는 확실한 차이 보임 Figure 3. Levels of CD5 on Naive CD8+ T Cells Correlate with the Strength of Responsiveness to Cytokines in Vitro and In Vivo CFSE-labeled B6 naïve CD5lo(Ly5.1) and CD5hi(Thy1.1) CD8+ T cells 6 days T cell-depleted hosts: irradated B6 or Rag1-/- >> T cell-depletion 상태에서 IL-7이 proliferation 시켰을 수 있음 (in vivo) (in vitro 상태에서는 IL-7에 반응이 적었음(Fig1)) >> 그렇다면, CD5hi T cells은 IL-7에 민감하게 반응하는가? SP, LN > CFSE analysis >>CD5hi T cell이 IL-7에 반응함 • CD5의 level과 cytokine에 대한 T cell의 responsiveness >> CD5hi T cells은 IL-2와 IL-7에 높은 반응성(hyperresponsiveness)을 보인다. 반면 CD5lo T cell은 두 cytokine에 반응을 적게 보인다. 그러나 TCR-CD3 ligation에 대한 반응에는 차이가 없다. >> 이 차이는 cytokine receptor의 수의 차이에 기인한 것일 수 있으나 CD122(IL-2Rb), CD127(IL-7Ra) 등에 차이가 적거나 거의 안 나타나는 것으로 볼 때 이 가능성은 희박하다. >> 또 다른 가설; IL-2의 binding으로 인해 IL-2R이 lipid rafts로 옮겨가 signal transduction 을 증가시키는 것이다. >> lipid rafts를 disruption 시킨다면? Figure 4. GM1 Expression on T Cell Subsets and the Effects of Disrupting Lipid Rafts on the Ability of Naive CD8+ T Cells to Respond to IL-2 inhibition of proliferation cholera toxin B staining GM1 detection CD4+ CD8+ CD4+ CD8+ Figure 4. GM1 Expression on T Cell Subsets and the Effects of Disrupting Lipid Rafts on the Ability of Naive CD8+ T Cells to Respond to IL-2 >> GM1 level에 따라 IL-2에 대한 반응이 CD5 level에 따른 반응과 유사 Figure 5. Expression of GM1 and CD5 on T Cell Subsets during Ontogeny HSA B6 or Tap1-/naïve B6 CD4+/CD8+ T cells(Ly5.1) 1 or 3 days SP, LN analysis from LN • T cell의 maturation 과정에서 GM1과 CD5의 expression level 변화 • DP에서 SP로 가는 과정에 CD5는 CD4+, CD8+ 모두에서 증가하지만 GM1은 SP CD8+에서만 크게 증가하는 것을 보임 • CD4+ T cell이 IL-2에 전혀 반응하지 않는 것은 높은 CD5, 낮은 GM1 level 과 관련이 있는 것으로 보임 Figure 6. Culturing Naive CD8+ T Cells with IL-2 Induces Lipid Raft Clustering and Colocalization of GM1 with IL-2Rβ IL-2R와 lipid rafts의 관계? ** 가설: IL-2가 lipid rafts로 옮겨가 signal transduction을 일으키는 것에 차이가 있다 CD5는 GM1이나 IL-2Rb와의 coclustering을 보이지 않았다.(data not shown) Fig S7 CD5lo/CD5hi T cells은 IL-2에 반응하여 signaling event에 차이를 보인다. • Naïve CD8 T cells이 cytokine에 강하게 반응하는 것은 높은 GM1 expression level과 lipid rafts, TCR-self MHC I ligand interaction에서 오는 signal들에 기인하는 것으로 볼 수 있다. • IL-2에 대한 CD8+ T cell의 hyperresponsiveness가 갖는 의미는 무엇인가? • CD8+ T cells이 antigen에 반응하기 위해서는 CD4+ T cells로부터 IL-2와 같은 “help”를 받아야 한다. strong antigen이나 TCR-CD3 ligation은(in vitro) CD8+ T cells 자신이 내는 IL-2가 충분하여 help없이도 proliferation 할 수 있게 한다. 그러나 weak antigen에 대해서는 CD8+ T cell이 IL-2를 충분히 내지 못해 exogenous IL-2가 있어야 proliferation 할 수 있다. • • • 따라서, weak antigen에 대한 반응은 IL-2에 대한 높은 반응을 보이는 cell에 의존적이라 할 수 있다. Figure 7. Hypersensitivity of CD8+ T Cells to IL-2 Augments Their Capacity to Respond to Foreign Antigens mimic weak Ag >> sol a-CD3 2C TCR(H-2b) – strong vs. weak ligand strong : endogenous (Balb/c Spl) H-2d highly immunogenic p2Ca weak : exogenous (B6 Spl) H-2b >> 전혀 proliferation이 일어나지 않아 OT-II와 함께 culture >> CD4로부터 IL-2 나와 proliferation 가 능해짐 Figure 7. Hypersensitivity of CD8+ T Cells to IL-2 Augments Their Capacity to Respond to Foreign Antigens Fig S8B IL-2 blockage에 의한 proliferation 감소 TCR과 antigen의 contact로 인해 IL-2에 대한 반 응이 증가하는 것은 lipid rafts가 새로이 합성되 어 증가하는 것과 관련이 있을 것이다 • The strong responsiveness of resting naïve CD8+ T cells to IL-2 was further enhanced by TCR contact with foreign antigens, thereby improving the immune response to both strong and weak antigens • CD5hi T cells give better response to strong or weak antigen than CD5lo T cells Conclusion & Discussion • • • • After positive selection in the thymus, continuous contact of naive CD8+ T cells with self-MHC-I ligands in the periphery induces covert TCR signals that promote sensitivity to several gc cytokines, including IL-7 and IL-2. Responsiveness to cytokines is most prominent for CD5hi T cells, i.e., cells with strong self reactivity, and correlates with high expression of GM1, implicating a role for lipid rafts. Sensitivity of naive CD8+ T cells to IL-2 becomes vital during the immune response. Thus, contact of CD8+ T cells with foreign antigen induces a further increase in cytokine sensitivity, thereby boosting the capacity of CD8+ T cells to receive help (IL-2) from CD4+ T cells. Further Study • TCR signal을 mimic 하여 T cell을 activation 시켰을 때 – CD99와 GM1의 양이 증가하는가 • FACS staining – 이때 CD99와 GM1은 colocalization을 보이는가 • confocal micorscopy – 이것은 T cell의 migration에 어떤 영향을 미치는가 • migration assay