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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: J Autoimmun. 2018 Jun 20;93:45–56. doi: 10.1016/j.jaut.2018.06.004

Figure 2. Adoptive Transfer Recipients of Germ-free CD25KO CD4+T cells Develop SS-like Disease.

Figure 2

CD4+ T cells were isolated from spleens and cervical lymph nodes (CLN) from either germfree (GF) CD25KO or conventional (CON) CD25KO mice and adoptively transferred (AT) i.p. into RAG1KO recipients (AT→ RAG). Ocular and lacrimal gland phenotype in RAG1KO recipients were investigated 5 weeks post-transfer.

(A) Corneal permeability measured as an uptake of fluorescent Oregon-Green dextran (OGD) dye. Bar graphs show means ± SEM of two independent experiments with eight to ten animals per experiment (final n = eighteen to twenty animals). Parametric t- tests were used to make comparisons between groups. (B). Representative pictures used to generate the bar graphs in A. (C) Number of PAS+ conjunctival goblet cells counted in paraffinembedded sections expressed as number per millimeter. Bar graphs show means ± SEM of two independent experiments with two to three animals per group, yielding a final sample of five right eyes for each group. Parametric t- tests were used to make comparisons between groups. (D) Inflammation scores of lacrimal gland pathology of donor and AT recipients. Bar graphs show average of two independent experiments (final n = thirteen right lacrimal glands animals). Nonparametric Mann–Whitney U test was used to make comparisons of inflammation scores. (E) Representative pictures of haematoxylin and eosin (H&E)-stained sections of lacrimal gland in adoptive transfer RAG1KO recipients; 20X objective, scale bar = 50 μm. (F) Flow cytometric analysis of intracellular staining of lacrimal gland and cervical lymph nodes of adoptive transfer recipients (n= eight to fourteen individual right lacrimal glands). Bar graphs show means ± SEM of three independent experiments. Parametric t-tests were used to make comparisons between groups. (G) Gene expression analysis in lacrimal gland lysates of germ-free and conventional CD25KO adoptive transfer recipients. (AT→ RAG). Bar graphs show means ± SD of two independent experiments (n = eight lacrimal glands/group).

*P < 0.05; ***P<0.001; ****P<0.0001 germ-free vs. conventional comparison

LG = lacrimal gland; CLN = cervical lymph nodes