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Our earlier immunoprofiling of the TLR7-agonistic imidazo-
quinolines had shown a very prominent activation of B- and
NK-cells, but minimal activation of T cells,7 and we asked if a pos-
sible reason could be differential uptake of the TLR7 agonist in
lymphocytic subsets. Flow cytometric analysis of the FITC-labeled
7 in experiments employing whole human blood indeed show a
prominent uptake of 7 in CD3ÀCD56+ NK and CD3ÀCD56À B lym-
phocytes as compared to CD3+CD56À T lymphocytes (Fig. 4).
The syntheses of fluorescent imidazoquinoline analogues that
retain TLR7-agonistic activity are expected to be useful probes in
examining their potential immunostimulatory and adjuvantic
properties.
5
10
B Lymphocytes
T Lymphocytes
NK Lymphocytes
4
3
10
10
10
2
1
10
4
5
3
2
1
10
CD56-APC
10
10
10
10
Acknowledgment
0
This work was supported by NIH/NIAID contract HHSN27
2200900033C.
0
1
2
3
4
5
Concentration of 7 (µg/mL)
Figure 4. Uptake of 7 in lymphocytic subsets as examined by flow cytometry.
Whole human blood was incubated with graded concentrations of 7 for 30 min,
lymphocytes stained with cell surface markers (anti-CD3-phycoerythrin [PE], and
anti-CD56-PE-allophycocyanin). Erythrocytes were lysed, and 105 total events were
acquired per sample.
Supplementary data
Supplementary data (experimental methods and characteriza-
tion of compounds (1H, 13C, and mass spectra)) associated with this
article can be found, in the online version, at doi:10.1016/
methyl)benzyl substituted analogue 5d was considerably more ac-
tive (EC50: 20 nM; Scheme 1) than its N1-(3-aminomethyl)benzyl
regioisomer 5c (EC50: 110 nM). The free primary amine on the N1
substituent of 5d was covalently coupled directly to commer-
cially-available fluorescein isothiocyanate and rhodamine B isothi-
ocyanate (Scheme 2). Conversely, the amine on 5d was converted
first to the isothiocyanate 6, allowing the subsequent coupling of
amine-bearing fluorophores, such as the bora-diazaindacene dye,
BODIPY-TR-cadaverine (Scheme 2). All three fluorescent conju-
gates retain TLR7-agonistic activity, although their potencies are
slightly attenuated relative to the parent compound, 5d (Fig. 2);
the EC50 values of 7, 8, and 9 are, respectively, 247 nM, 115 nM,
and 108 nM.
Incubation of murine macrophage J774.A1 cells with 8 or 9, fol-
lowed by intravital epi- and confocal fluorescence microcopy
showed prominent perinuclear localization, which is consistent
with the expected endosomal distribution of TLR7.14 Shown in
Figure 3 is a representative epifluorescence micrograph of J774
cells treated with 9 at 100 nM concentration.
References and notes
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