Download 100313-Immunity-Lipid raft T cell homeostasis.[281]

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