Figure 3. FMNL1-deficient T cells are impaired in trafficking to the CNS and inducing EAE.

(A) Experimental set-up to quantify activated T cell trafficking to the inflamed CNS. Activated, differentially dye-labeled, polyclonal CD45.2/.2 WT and FMNL1 KO T cells were co-transferred intravenously at a 1:1 ratio into CD45.1/.1 mice with ongoing EAE (score ≥2.0) and T cell trafficking to the indicated tissues was quantified by flow cytometry 24 hr post-transfer. (B) FMNL1-deficient T cells are impaired in trafficking to the inflamed CNS. Ratio of co-transferred FMNL1 KO to WT T cells in the indicated tissues (left). Values were normalized to the FMNL1 KO:WT ratio in the injected sample. A ratio below 1.0 (dashed line) indicates impaired trafficking to the tissue. Number of transferred WT or FMNL1 KO T cells recovered from the indicated tissues (right). (C) Representative spinal cord and blood flow plots and gating strategy for identifying co-transferred dye-labeled T cells quantified in B. (D) Experimental set up for induction of EAE via T cell transfer. Control or FMNL1 KO MOG-specific 2D2 T cells were activated ex vivo and then transferred into WT recipient mice. EAE disease severity was scored daily for 28 days. (E) FMNL1 deficiency in T cells delays EAE onset and partially protects from disease. EAE incidence (score ≥1.0) in mice receiving control or FMNLL1 KO 2D2 T cells (left). Mean EAE score ± SEM over time in mice receiving control or FMNL1 T cells 2D2 T cells (right). Data in B are the mean ± SEM (left) or individual means (right) from 4 independent experiments with 2 recipient mice per group; data in E are pooled from 3 independent experiments with cohorts of 5 mice/group each. Statistics in B were calculated using a one-sample two-tailed t-test against a theoretical FMNL1 KO:WT ratio of 1.0 (left) or repeated measures one-way ANOVA with Sidak’s multiple comparisons test (right); statistics in E (left) were calculated by Log-rank test; statistical interaction of genotype with disease severity over time in E (right) was calculated by repeated measures two-way ANOVA. n.s. = not significant, *=p < 0.05, **=p < 0.01.

Figure 3—figure supplement 1. FMNL1 deficiency does not impair ex vivo T cell cognate peptide stimulation.

CD4⁺ T cells from WT or FMNL1 KO 2D2 TCR transgenic mice were isolated and then activated ex-vivo with MOG peptide in the presence of WT irradiated, congenically marked, CD45.1⁺ splenocytes. After 2 days, T cells were cultured with IL-2 for 4 days. (A) WT and FMNL1 KO T cells proliferate equivalently in response to cognate peptide stimulus. Total number of WT or KO T cells on days 0, 2, 4 and 6 days post stimulus. (B) Cognate peptide activated WT and FMNL1 KO T cells express equivalent levels of adhesion molecules and activation markers. Ratio of geometric mean fluorescent intensity (gMFI) of antibody staining of indicated surface molecules of FMNL1 KO T cells compared to WT T cells 7 days after activation. A ratio above or below 1.0 (dashed line) indicates a respective increase or decrease in expression of the indicated marker on FMNL1 KO T cells relative to WT. Data in A and B are the mean ± SEM from 3 independent experiments. Statistics in A were calculated using repeated measures two-way ANOVA with Sidak’s multiple comparisons test; statistics in B were calculated using a one-sample two-tailed t-test against a theoretical FMNL1 KO:WT gMFI ratio of 1.0. n.s. = not significant.

Figure 3—figure supplement 2. FMNL1 deficiency does not impair T cell trafficking to lymphoid tissues.

(A) Experimental set-up to quantify naive T cell trafficking to lymphoid organs. Differentially fluorescent dye-labeled naive WT and FMNL1 KO T cells were co-transferred intravenously at a 1:1 ratio into CD45.1/.1 recipient mice and T cell trafficking to lymphoid tissues was quantified by flow cytometry 24 hr post-transfer. (B) Representative lymph node flow plots and gating strategy for identifying co-transferred dye-labeled T cells. (C) Ratio of co-transferred FMNL1 KO to WT T cells in the indicated tissues (left). Values were normalized to the FMNL1 KO:WT ratio in the injected sample. A ratio below 1.0 (dashed line) indicates impaired trafficking to the tissue. Number of transferred WT or FMNL1 KO T cells recovered from the indicated tissues (right). (D) Experimental set-up to quantify activated T cell trafficking to lymphoid organs. Differentially dye-labeled activated WT and FMNL1 KO T cells were co-transferred intravenously at a 1:1 ratio into CD45.1/.1 recipient mice and T cell trafficking to lymphoid tissues was quantified by flow cytometry 24 hr post transfer. (E) Ratio of co-transferred FMNL1 KO to WT T cells in the indicated tissues (left). Values were normalized to the FMNL1 KO:WT ratio in the injected sample. A ratio below 1.0 (dashed line) indicates impaired trafficking to the tissue. Number of transferred WT or FMNL1 KO T cells recovered from the indicated tissues (right). Data in C are the mean ± SEM (left) or individual means (right) from 3 independent experiments with 2 recipient mice each per experiment. Data in E are the mean ± SEM (left) or individual means (right) from 4 independent experiments with 2 recipient mice each per experiment. Statistics in E and C were calculated using a one-sample two-tailed t-test against a theoretical FMNL1 KO:WT ratio of 1.0 (left) or by repeated measures one-way ANOVA with correction with Sidak’s multiple comparisons test (right). n.s. = not significant, *=p < 0.05.