Lipid length controls antigen entry into endosomal and nonendosomal pathways for CD1b presentation

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Lipid length controls antigen entry into
endosomal and nonendosomal pathways for CD1b
presentation
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CD1 Proteins

• CD1 is a conserved family of non-(MHC) encoded
  MHC like Glycoproteins that specialize in
  presenting lipids
• Four human CD1 proteins CD1A, CD1B, CD1C and
  CD1D present lipid antigens by insertion of
  lipids into a groove in the proteins to form
  CD1lipid complexes.
• These complexes activate T cells after direct
  recognition by specific T-cell receptors.

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Where are CD1 proteins found?

• Cortical Thymocytes
• carry out positive selection of CD1 restricted T
  cells.
• Antigen Presenting Cells
• Myeloid dendritic cells
• Macrophages
• B cells

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Structure of human CD1B
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Figure 1. CD1b-mediated T cell activation by GMM
antigens of varying alkyl chain length. (a) The
positive mode electrospray ionization mass
spectra (ESI-MS) of glucose monomycolate isolated
from M. phlei, N. farcinica and R. equi showed
ions corresponding to an alkane series of sodium
adducts of GMM with the indicated lipid length
Cx. (b) Synthetic condensation of free fatty
acids of varying length yielded mycolic acids
(Cx) with an   -branch (Cx/2-2) and a
meromycolate (mero) chain (Cx/22) that were
glucosylated as described20, 28. Positive mode
ESI-MS of synthetic GMM with a C32 mycolic acid
is shown, and the lengths of the   - and
meromycolate chains of the other analogs are
summarized. (c,d) The proliferative responses of
the CD1b-restricted T cell line LDN5 to GMMs of
the indicated lipid lengths were measured by
3Hthymidine incorporation. Data are mean
   s.d. of triplicate samples.
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Figure 2. DCs internalized GMM antigens and
selectively delivered C80 GMM to late endosomal
and lysosomal compartments. (a) Biosynthetically
labeled 14C C32 GMM was cultured with DCs in
the presence or absence of metabolic inhibitors
(0.02 NaN3 and 50 mM 2-DOG). Cells were
extensively washed with media, then dissolved in
scintillant to measure cell-associated GMM. (b)
Metabolism-dependent association (cellular
uptake) of C32 and C80 GMM with DCs was measured
at 0.5  g/ml. (c) DCs (105) were cultured with
14CC80 or 14CC32 GMM (7.5  g/ml) for 24 h
and washed. The GMM-pulsed DCs were disrupted by
shearing, centrifuged to remove nuclei and
fractionated by density centrifugation on Percoll
gradients as described30. Gradient fractions
(110) were assayed for   -hexosaminidase
activity by ELISA and for distribution of CD1b
and markers of early endosomes and plasma
membrane (Rab5, transferrin receptor and MHC
class I) by immunoblotting30. (d) (Upper panel)
C32 and C80 GMM content in gradient fractions was
detected by scintillation counting, which shows
selective accumulation of C80 GMM in dense
fractions (13). One representative of four
experiments is shown. (Lower panel) The
calculated ratios of C80 versus C32 GMM in each
fraction compared to the distribution of
  -hexosaminidase for the pooled data from all
four experiments.
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Figure 3. B lymphoblastoid cells and DCs differed
in the efficiency of uptake and presentation of
GMM alkyl chain length analogs. (a) Irradiated
CD1b-transfected C1R cells or DCs (105
cells/well) were cultured with GMMs of the
indicated chain lengths, PMA (10 ng/ml) and
J.RT3/LDN5       cells. After 24 h,
supernatants were tested for IL-2 production by
measuring 3Hthymidine incorporation into
IL-2dependent HT-2 cells. (b) Uptake of C32 GMM
(0.2  g/ml) by DCs and C1R cells was measured
as in Fig. 2. (c) Irradiated DCs were fixed with
0.02 glutaraldehyde before or after 4 h of
culture with GMM as described21. The
proliferative response of LDN5 T cells after 4
days of culture with antigen-pulsed DCs was
measured by 3Hthymidine incorporation. (Ag,
antigen.)
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Figure 4. Presentation of GMMs with short alkyl
chains was more rapid, but less stable, than
presentation of GMMs with longer alkyl chains.
(a) Resting LDN5 T cells were loaded with Fura
red and Fluo-4 dyes and subjected to flow
cytometric analysis. Release of intracellular
calcium after treatment with 1  g/ml of
ionomycin (MFI317 in FL1) or coincubation with
C1R cells loaded with 20  M C80 GMM (MFI239 in
FL1) for 4 h were measured by the increase in
fluorescence intensity at low wavelengths (FL1)
and inhibition of fluorescence intensity at
higher wavelengths (FL3). Data are expressed as
the percentage of dye-treated cells within the
high FL1, low FL3 gate. (b) Comparison of LDN5
activation by ionomycin (iono), C1R.CD1b cells
(CD1b) or C1R.mock cells (mock) that were treated
with 20  M C32 GMM (C32) or C80 GMM (C80) for
20 h. Data were collected for 20 s or 5000 events
after centrifugation (spin), or not, of cells for
60 s. (c) C1R.CD1b cells were preincubated with
20  M C80 GMM or C32 GMM for the indicated
times, added at a 11 ratio LDN5 cells,
centrifuged for 60 s and immediately assayed for
calcium flux. (d) C1R.CD1b cells were pulsed with
C32 GMM or C80 GMM at the indicated
concentrations for either 20 h or 30 min. After
the antigen pulse (P, prewash), cells were
extensively washed, chased in antigen-free medium
for the indicated times, then tested for antigen
presentation by exposing them to LDN5 T cells and
immediately assaying these for calcium flux.
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Figure 5. Presentation of GMM chain length
analogs by cells expressing CD1b proteins that
lack endosomal targeting sequences. (ac) IL-2
release by J.RT-3/LDN5       cells was measured
in response to GMM of the indicated alkyl chain
lengths presented by C1R cells expressing
wild-type CD1b (WT), CD1b.TD (TD), CD1b.Y311A
(Y311A) or CD1b.DAF (DAF)22, 35. The cell surface
expression of CD1b by clones was evaluated by
flow cytometric analysis with the murine mAb 4A7
MFIs are given in parentheses. C1R clones
expressing CD1b, CD1b.TD and CD1b.Y311A cells
that expressed equivalent MFIs were chosen for
analysis. (d) IL-2 release by J.RT-3 cells was
measured in response to C32 GMM treated C1R
clones that expressed low medium or high amounts
of CD1b or CD1b.TD. C1R.mock cells were used as a
control.
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Figure 6. Transformed thymocytes more efficiently
presented C32 GMM. IL-2 release by JRT-3/LDN5   
   cells in response to C32 GMM (open) and C80
GMM (filled) presented by DCs or HPB-ALL cells
was measured as in Fig. 3.
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