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. 2022 May 4;5:413. doi: 10.1038/s42003-022-03344-6

Fig. 2. ICOS+ T cells regulate Ltα expression within newly formed TLSs.

Fig. 2

a Representative dot plots showing flow cytometry staining for ICOS expression in wt salivary glands at day 5 p.c. by CD45+ cells. Graph showing percentage of ICOS+ T lymphocytes and NK cells infiltrating salivary glands at day 5 p.c. b viSNE plots of ICOS+ T lymphocytes from infected salivary gland from wt mice at day 5 p.c., analyzed by multicolor flow cytometry. Colours indicate cell expression level of labelled markers (LTα3 and CD4). c, d Flow cytometry analysis of absolute numbers of T cells and LTα-producing T cells in wt (black bars) and Icosl−/− (white bars) mice at day 5 p.c. e, f qPCR analysis of ltα and icos mRNA transcripts in FACS sorted CD3+ cells from wt (black bars) and Icosl−/− (white bars) mice at day 5 p.c. mRNA transcripts were normalized to housekeeping gene β-actin and presented as RQ values calculated with calibrator day 0 CD45+ cells. g wt mice were left untreated or treated with anti-ICOS blocking antibody from day 0 post salivary gland cannulation and sacrificed at days 5 and 8 p.c. Cannulated Icosl−/− were also sacrificed and analyzed at days 5 and 8 p.c. Representative microphotographs of salivary glands examined by immunofluorescence for CD3ε (red) and CD19 (blue) showing lymphocytic infiltration. Scale bar 100 µm. h qPCR analysis of mRNA obtained from salivary glands at day 5 and 8 p.c. of lymphoid chemokine ccl19 and cxcl13 in anti-ICOS treated wt mice or Icosl−/− mice (both white bars) in comparison to wt mice (black bars). mRNA transcripts were normalized to β-actin mRNA and results are presented as relative quantitation (RQ). i Transcript levels for ccl19, ccl21c and cxcl13 in FACS sorted CD45-EpCAM-CD31-pdpn+ immunofibroblasts from wt (black bars) and Icosl−/− (white bars) mice at day 5 p.c. Transcripts levels were normalised to β-actin. Data are mean ± s.e.m from two independent experiments with three to six mice analyzed per group. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. non-significant, unpaired t test.