Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A and B ) Tumor volume in Flot2 +/+ or Flot2 –/– mice injected with B16F10 ( A ) or MC38 ( B ) ( n = 6–7 per group). ( C – J ) Flow cytometric analysis of tumor-infiltrating lymphocytes (TILs) in B16F10 tumor–bearing Flot2 +/+ or Flot2 –/– mice. Representative plots ( C , E , and H ) are shown. TCRβ + ( D ), TCRβ + CD4 + ( F ), and TCRβ + CD8 + ( G ) populations within 7AAD – CD45 + population, and Ki67 + populations among 7AAD – CD45 + TCRβ + CD4 + CD44 + CD62L – population ( I ) or 7AAD – CD45 + TCRβ + CD8 + CD44 + CD62L – population ( J ), are depicted. ( K ) Splenocytes from B16F10 tumor–bearing Flot2 +/+ or Flot2 –/– mice were stimulated with 1 μg/mL of TRP-2 melanoma peptide for 24 hours and assayed for antigen-specific reactivity using an IFN-γ ELISPOT assay. Data are representative of 2 independent experiments. Data were analyzed by unpaired t test ( D , F , G , and I – K ) or 2-way ANOVA followed with Šidák’s multiple-comparison tests ( A and B ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: Injection, Enzyme-linked Immunospot, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A and B ) Tumor volume in Flot2 WT or Flot2 CD4 mice injected with B16F10 ( A ; n = 10 per group) or MC38 ( B , n = 9 for Flot2 WT and n = 14 for Flot2 CD4 ). ( C – H ) Flow cytometric analysis of TILs. Representative plots ( C and F ) are shown. TCRβ + CD4 + ( D ) and TCRβ + CD8 + ( E ) populations among 7AAD – CD45.2 + population and Ki67 + populations among 7AAD – CD45.2 + TCRβ + CD4 + population ( G ) or 7AAD – CD45.2 + TCRβ + CD8 + population ( H ) in B16F10-bearing Flot2 WT or Flot2 CD4 mice are presented. ( I – N ) Flow cytometric analysis of tumor-draining lymph nodes (dLNs). Representative plots ( I and L ) are displayed. CD44 + IFN-γ + ( J ) and CD44 + TNF-α + ( K ) populations among 7AAD – CD45.2 + TCRβ + CD4 + population and CD44 + IFN-γ + ( M ) and CD44 + TNF-α + ( N ) among 7AAD – CD45.2 + TCRβ + CD8 + population in B16F10-bearing Flot2 WT or Flot2 CD4 mice are indicated. Data are representative of 2 independent experiments ( A – N ). Data were analyzed by unpaired t test ( D , E , G , H , J , K , M , and N ) or 2-way ANOVA followed with Šidák’s multiple-comparison tests ( A and B ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: Injection, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A ) Weight loss in Flot2 +/+ or Flot2 –/– mice following Listeria monocytogenes infection (5,000 CFU per mouse) is presented as the mean percentage of initial weight ( n = 4–5 per group). ( B – G ) Flow cytometric analysis of splenic T cells from Listeria -infected Flot2 +/+ or Flot2 –/– mice. Representative plots ( B and E ) are provided. CD44 + Ki67 + ( C ) and TNF-α + ( D ) populations within viable CD45 + TCRβ + CD4 + population and CD44 + Ki67 + ( F ) and TNF-α + ( G ) populations within viable CD45 + TCRβ + CD8 + population are shown. ( H ) Weight loss in Flot2 WT or Flot2 CD4 mice following L. monocytogenes infection (5,000 CFU per mouse) is presented as the mean percentage of initial weight ( n = 5–6 per group). ( I ) Splenic CD4 + and CD8 + T cells numbers in infected Flot2 WT or Flot2 CD4 mice are depicted. ( J – Q ) Flow cytometric analysis of splenic T cells from L. monocytogenes –infected Flot2 WT or Flot2 CD4 mice. Representative plots ( J , L, and O ) are shown. TNF-α + IFN-γ + IL-2 + ( K ), CD44 + Ki67 + ( M ), and TNF-α + ( N ) populations within viable CD45 + TCRβ + CD4 + population and CD44 + Ki67 + ( P ) and TNF-α + ( Q ) populations within viable CD45 + TCRβ + CD8 + population are shown. Data are representative of 2 independent experiments ( A – Q ). Data were analyzed by unpaired t test ( C , D , F, G, I , K , M , N , P , and Q ) or 2-way ANOVA followed with Šidák’s multiple-comparison tests ( A and H ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: Infection, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A – C ) Naive CD4 + T cells were stimulated in vitro for 3 hours ( B ) or 24 hours ( C ) with varying doses of plate-bound αCD3, alongside a fixed dose of soluble αCD28 (1 μg/mL), followed by FACS. Representative plots ( A ) show Nur77 + ( B ) and CD69 + ( C ) populations within viable TCRβ + CD4 + population. ( D – G ) Western blot of the phosphorylation of TCR signaling molecules in naive CD4 + T cells stimulated with varying doses of plate-bound αCD3 for 3 minutes. Representative blots ( D ) and quantifications of pZAP70 ( E ), pERK1/2 ( F ), and pLck (Y505) ( G ), normalized to their respective total protein levels (ZAP70, ERK1/2, and Lck), are shown. ( H and I ) scRNA-Seq was performed on naive CD4 + T cells following a 3-hour stimulation with varying concentrations of plate-bound αCD3. A fixed dose of soluble αCD28 (1 μg/mL) was provided under the conditions of 0.25 or 1 μg/mL of plate-bound αCD3. Unsupervised T cell clusters were annotated as 5 distinguishable functional states on a UMAP plot ( H ), and the effect of stimulation dose was projected onto the UMAP ( I ). ( J ) Distribution of Flot2 WT or Flot2 CD4 CD4 + T cells in each cluster. Red arrows indicate the priming clusters for each genotype. ( K ) Occupancy of Flot2 WT or Flot2 CD4 genotype in each T cell cluster, analyzed by total or each stimulatory condition. ( L ) Volcano plots of spliced RNA in naive, priming, and activated clusters. ( M ) Expression level of spliced RNA related to cellular proliferation in the naive cluster. Data are pooled from 3 ( A – C ), 5 ( E ), 8 ( F ), or 7 ( G ) independent experiments. Data were analyzed by 1-way ANOVA followed with Šidák’s multiple-comparison tests ( B , C , and E – G ), χ 2 analysis ( K ), or Wilcoxon ranked-sum test ( M ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: In Vitro, Western Blot, Phospho-proteomics, Functional Assay, Expressing, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A – H ) dSTORM analysis of TCR nanoclustering in Flot2 WT and Flot2 CD4 naive CD4 + T cells. Clustering images of CD3ε molecules ( A and B ) or TCRβ molecules ( E and F ) are shown. Number of clusters and voxel of CD3ε ( C and D ) or TCRβ ( G and H ) molecules after quantification using Huygens Cluster Analyzer are depicted. Scale bars: 1 μm. Data are representative of 4 ( A – D ) or 2 ( E – H ) independent experiments. Data were analyzed by unpaired t test ( C , D , G , and H ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques:

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A and B ) Tumor volume in Flot2 +/+ or Flot2 –/– mice injected with B16F10 ( A ) or MC38 ( B ) ( n = 6–7 per group). ( C – J ) Flow cytometric analysis of tumor-infiltrating lymphocytes (TILs) in B16F10 tumor–bearing Flot2 +/+ or Flot2 –/– mice. Representative plots ( C , E , and H ) are shown. TCRβ + ( D ), TCRβ + CD4 + ( F ), and TCRβ + CD8 + ( G ) populations within 7AAD – CD45 + population, and Ki67 + populations among 7AAD – CD45 + TCRβ + CD4 + CD44 + CD62L – population ( I ) or 7AAD – CD45 + TCRβ + CD8 + CD44 + CD62L – population ( J ), are depicted. ( K ) Splenocytes from B16F10 tumor–bearing Flot2 +/+ or Flot2 –/– mice were stimulated with 1 μg/mL of TRP-2 melanoma peptide for 24 hours and assayed for antigen-specific reactivity using an IFN-γ ELISPOT assay. Data are representative of 2 independent experiments. Data were analyzed by unpaired t test ( D , F , G , and I – K ) or 2-way ANOVA followed with Šidák’s multiple-comparison tests ( A and B ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: Injection, Enzyme-linked Immunospot, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A and B ) Tumor volume in Flot2 WT or Flot2 CD4 mice injected with B16F10 ( A ; n = 10 per group) or MC38 ( B , n = 9 for Flot2 WT and n = 14 for Flot2 CD4 ). ( C – H ) Flow cytometric analysis of TILs. Representative plots ( C and F ) are shown. TCRβ + CD4 + ( D ) and TCRβ + CD8 + ( E ) populations among 7AAD – CD45.2 + population and Ki67 + populations among 7AAD – CD45.2 + TCRβ + CD4 + population ( G ) or 7AAD – CD45.2 + TCRβ + CD8 + population ( H ) in B16F10-bearing Flot2 WT or Flot2 CD4 mice are presented. ( I – N ) Flow cytometric analysis of tumor-draining lymph nodes (dLNs). Representative plots ( I and L ) are displayed. CD44 + IFN-γ + ( J ) and CD44 + TNF-α + ( K ) populations among 7AAD – CD45.2 + TCRβ + CD4 + population and CD44 + IFN-γ + ( M ) and CD44 + TNF-α + ( N ) among 7AAD – CD45.2 + TCRβ + CD8 + population in B16F10-bearing Flot2 WT or Flot2 CD4 mice are indicated. Data are representative of 2 independent experiments ( A – N ). Data were analyzed by unpaired t test ( D , E , G , H , J , K , M , and N ) or 2-way ANOVA followed with Šidák’s multiple-comparison tests ( A and B ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: Injection, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A ) Weight loss in Flot2 +/+ or Flot2 –/– mice following Listeria monocytogenes infection (5,000 CFU per mouse) is presented as the mean percentage of initial weight ( n = 4–5 per group). ( B – G ) Flow cytometric analysis of splenic T cells from Listeria -infected Flot2 +/+ or Flot2 –/– mice. Representative plots ( B and E ) are provided. CD44 + Ki67 + ( C ) and TNF-α + ( D ) populations within viable CD45 + TCRβ + CD4 + population and CD44 + Ki67 + ( F ) and TNF-α + ( G ) populations within viable CD45 + TCRβ + CD8 + population are shown. ( H ) Weight loss in Flot2 WT or Flot2 CD4 mice following L. monocytogenes infection (5,000 CFU per mouse) is presented as the mean percentage of initial weight ( n = 5–6 per group). ( I ) Splenic CD4 + and CD8 + T cells numbers in infected Flot2 WT or Flot2 CD4 mice are depicted. ( J – Q ) Flow cytometric analysis of splenic T cells from L. monocytogenes –infected Flot2 WT or Flot2 CD4 mice. Representative plots ( J , L, and O ) are shown. TNF-α + IFN-γ + IL-2 + ( K ), CD44 + Ki67 + ( M ), and TNF-α + ( N ) populations within viable CD45 + TCRβ + CD4 + population and CD44 + Ki67 + ( P ) and TNF-α + ( Q ) populations within viable CD45 + TCRβ + CD8 + population are shown. Data are representative of 2 independent experiments ( A – Q ). Data were analyzed by unpaired t test ( C , D , F, G, I , K , M , N , P , and Q ) or 2-way ANOVA followed with Šidák’s multiple-comparison tests ( A and H ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: Infection, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A – C ) Naive CD4 + T cells were stimulated in vitro for 3 hours ( B ) or 24 hours ( C ) with varying doses of plate-bound αCD3, alongside a fixed dose of soluble αCD28 (1 μg/mL), followed by FACS. Representative plots ( A ) show Nur77 + ( B ) and CD69 + ( C ) populations within viable TCRβ + CD4 + population. ( D – G ) Western blot of the phosphorylation of TCR signaling molecules in naive CD4 + T cells stimulated with varying doses of plate-bound αCD3 for 3 minutes. Representative blots ( D ) and quantifications of pZAP70 ( E ), pERK1/2 ( F ), and pLck (Y505) ( G ), normalized to their respective total protein levels (ZAP70, ERK1/2, and Lck), are shown. ( H and I ) scRNA-Seq was performed on naive CD4 + T cells following a 3-hour stimulation with varying concentrations of plate-bound αCD3. A fixed dose of soluble αCD28 (1 μg/mL) was provided under the conditions of 0.25 or 1 μg/mL of plate-bound αCD3. Unsupervised T cell clusters were annotated as 5 distinguishable functional states on a UMAP plot ( H ), and the effect of stimulation dose was projected onto the UMAP ( I ). ( J ) Distribution of Flot2 WT or Flot2 CD4 CD4 + T cells in each cluster. Red arrows indicate the priming clusters for each genotype. ( K ) Occupancy of Flot2 WT or Flot2 CD4 genotype in each T cell cluster, analyzed by total or each stimulatory condition. ( L ) Volcano plots of spliced RNA in naive, priming, and activated clusters. ( M ) Expression level of spliced RNA related to cellular proliferation in the naive cluster. Data are pooled from 3 ( A – C ), 5 ( E ), 8 ( F ), or 7 ( G ) independent experiments. Data were analyzed by 1-way ANOVA followed with Šidák’s multiple-comparison tests ( B , C , and E – G ), χ 2 analysis ( K ), or Wilcoxon ranked-sum test ( M ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: In Vitro, Western Blot, Phospho-proteomics, Functional Assay, Expressing, Comparison

Journal: JCI Insight

Article Title: Flotillin-2 dampens T cell antigen sensitivity and functionality

doi: 10.1172/jci.insight.182328

Figure Lengend Snippet: ( A – H ) dSTORM analysis of TCR nanoclustering in Flot2 WT and Flot2 CD4 naive CD4 + T cells. Clustering images of CD3ε molecules ( A and B ) or TCRβ molecules ( E and F ) are shown. Number of clusters and voxel of CD3ε ( C and D ) or TCRβ ( G and H ) molecules after quantification using Huygens Cluster Analyzer are depicted. Scale bars: 1 μm. Data are representative of 4 ( A – D ) or 2 ( E – H ) independent experiments. Data were analyzed by unpaired t test ( C , D , G , and H ). Data are shown as mean ± SEM; * P < 0.05; ** P < 0.01.

Article Snippet: Total RNA was obtained using RNeasy kits (Qiagen) following manufacturer instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). cDNA was quantified with TaqMan Universal PCR Master Mix (Invitrogen) and predesigned TaqMan primers (Assay ID: Mm00514962_g1, Mm01241315_g1; both Flot2).

Techniques: