NKT Cell Stimulation with α-Galactosylceramide Results in a Block of Th17 Differentiation after Intranasal Immunization in Mice

In a previous study we demonstrated that intranasal (i.n.) vaccination promotes a Th17 biased immune response. Here, we show that co-administration of a pegylated derivative of α-galactosylceramide (αGCPEG) with an antigen, even in the presence of Th17-polarizing compounds, results in a strong blocking of Th17 differentiation. Additional studies demonstrated that this phenomenon is specifically dependent on soluble factors, like IL-4 and IFNγ, which are produced by NKT cells. Even NK1.1 negative NKT cells, which by themselves produce IL-17A, are able to block Th17 differentiation. It follows that the use of αGCPEG as adjuvant would enable to tailor Th17 responses, according to the specific clinical needs. This knowledge expands our understanding of the role played by NKT cells in overall control of the cytokine microenvironment, as well as in the overall shaping of adaptive immune responses

αGCPEG efficiently modulates the effect of other adjuvants.

<p>C57BL/6 mice were immunized with OVA co-administered with different combinations of LPS, Curdlan and αGCPEG. Control animals received PBS or OVA alone. After 2 boosts splenocytes were isolated from animals and cells (5×10⁵/well) were then cultured in ELISpot plates coated with anti-IL-17A antibody for 48 h. Subsequently, plates were incubated with detection antibody, developed and the numbers of spots were counted. From the presented data the background was subtracted. *, statistically significant (p<0.05). Results belong to one representative out of 3 independent experiments.</p

NKT cells block Th17 differentiation <i>in vitro</i> by soluble factors.

<p>Naïve CD4⁺ cells were sorted from spleen of OTII animals and stained with CFSE. Cells were then co-cultured with DC under Th17 inducing conditions together with a peptide encompassing the specifically recognized epitope (OVA323–339) in the presence of either αGCPEG or BPPcysPEG. After 4 days in culture the cells were stained for IL-17A production and analyzed by FACS. (A) To some cultures NKT cells were added (right panels). (B) To some cultures were added supernatants collected from 24 h cultures of DC in presence of αGCPEG (left panel) or co-cultures of DC and NKT cells in the presence of αGCPEG (right panel). Results belong to one representative out of at least 3 independent experiments.</p

NK1.1⁻ NKT cells also block Th17 differentiation.

<p>Naïve CD4⁺ cells were sorted from spleen of OTII animals and stained with CFSE. Cells were then co-cultured with DC under Th17 inducing conditions with the specific peptide (OVA323–339) and αGCPEG in the absence (left panels) or in the presence of either NK1.1⁺ (central panels) or NK1.1⁻ (right panels) NKT cells. Some cultures also received blocking antibodies against IL-4, IL-10 or IFNγ. After 4 days in culture, cells were stained for IL-17A production and analyzed by FACS. Results belong to one representative out of 3 independent experiments.</p

αGCPEG blocks induction of Th17 immune responses after i.n. immunization.

<p>C57BL/6 mice were immunized with OVA co-administered with different adjuvants. Control animals received PBS or OVA alone. After 2 boosts on day 14 and 21, splenocytes were isolated and cells were then cultured in ELISpot plates coated with anti-IL-17A antibody for 48 h. Subsequently, plates were incubated with detection antibody, developed and the numbers of spots were counted. From the presented data the background was subtracted. *, statistically significant (p<0.05). Results belong to one representative out of 3 independent experiments.</p

NKT cells block Th17 differentiation by secretion of IL-4 and IFNγ.

<p>(A) Naïve CD4⁺ cells were sorted from spleens of OTII animals. Cells were then co-cultured with DC under Th17 inducing conditions with the specific peptide (OVA323–339) under stimulation with either αGCPEG or BPPcysPEG in the presence or absence of NKT cells. After 24 h culture supernatants were collected and the levels of IL-4, IFNγ and IL-2 were measured by ELISA. (B) Naïve CD4⁺ cells were sorted from spleen of OTII animals and stained with CFSE. Cells were then co-cultured with DC under Th17 inducing conditions with the specific peptide (OVA323–339) and αGCPEG, in the presence or absence of NKT cells. To some cultures neutralizing antibodies against IL-4 and/or IFNγ were added. After 4 days of culture the cells were stained for IL-17A production and analyzed by FACS. Results belong to one representative out of at least 3 independent experiments.</p

NKT cells block Th17 differentiation after i.n. immunization <i>in vivo</i>.

<p>C57BL/6 and Jα281 KO mice were immunized with OVA co-administered with αGCPEG. Control animals received PBS, OVA or OVA with the TLR2/6 agonist BPPcysPEG. After 1 boost on day 14, splenocytes were isolated and cells (1×10⁶/well) were then cultured in ELISpot plates coated with anti-IL-17A antibodies for 48 h. Subsequently, plates were incubated with detection antibodies, developed and the numbers of spots were counted. From the presented data the background was subtracted. *, statistically significant (p<0.05). Results belong to one representative out of 2 independent experiments.</p