Figure 6. ILC1s in the draining LN are a rapid source of IFN-γ after immunization.

To investigate the function of ILC1 within peripheral LNs, mice were injected intramuscularly with 20 μg OVA-2W1S/PBS or 20 μg OVA-2W1S/AS01 and culled either 6 hrs post immunization to assess the early innate IFN-γ response or after 7 days following a second immunization performed 6 hrs prior to analysis. (A) Representative flow cytometry plots showing inguinal LN cells from mice given OVA-2W1S/PBS (top) or OVA-2W1S/AS01 (bottom), with identification of different IFN-γ producing populations including cNK cells (CD45⁺ IFN-γ⁺ CD3ε^- NK1.1⁺EOMES⁺) and ILC1s (CD45⁺ IFNγ⁺ CD3ε^- NK1.1⁺ EOMES^- T-bet⁺) shown. (B) Total number of IFN-γ⁺ ILC1s and cNK cells in OVA/ASO1 treated mice (n=5). (C) Representative flow cytometry plots showing IFN-γ production by 2W1S-specific CD4⁺ T cells at day 7 post initial immunization with (AS01) or without (PBS) the adjuvant AS01. (D) Total number of IFN-γ producing 2W1S-specific CD4 T cells in the draining inguinal LN of PBS (n=4) and AS01 mice (n=6). To test whether both migratory and resident ILC1 subsets were able to produce IFN-γ, the bLN of Kaede mice was photoconverted and 3 days later mice were then immunized in the paw pad with either 20 μg OVA-2W1S/PBS or 20 μg OVA-2W1S/AS01 and analyzed 6 hrs later. (E) Representative flow cytometry plots showing bLN cells from mice given OVA-2W1S/PBS (top) or OVA-2W1S/AS01 (bottom), with identification of ILC1 and cNK populations producing IFN-γ and their expression of Kaede Green versus Kaede Red protein. (F) Percentage Kaede Green (migratory) and Kaede Red (resident) ILC1s and cNK cells amongst IFN-γ producing cells. (G) Total numbers of IFN-γ⁺ ILC1s and cNK cells expressing either Kaede Green (migratory) or Kaede Red (resident). All sets of data are pooled from 2 independent experiments. Values on flow cytometry plots represent percentages, bars on scatter plots represents the median, which is also shown numerically. Statistical significance was tested using an unpaired, non-parametric, Mann-Whitney two-tailed U test: *p≤0.05, **p≤0.01, ***p≤0.001.