Journal: bioRxiv

Article Title: TCR/ITK signaling via mTOR tunes CD8 + T cell homeostatic proliferation, metabolism, and anti-tumor effector function

doi: 10.1101/359604

Figure Lengend Snippet: (A) Representative plots of expression of CD44/CD122 on splenic CD8 + T cell from non-transgenic (Non-Tg) or OTI- Rag −/− transgenic WT and Itk −/− mice (haplotype H2K b ). (B) Experimental model of WT and Itk −/− CD8 + T cell expansion under lymphopenic environments. Naïve CD8 + T cells were flow-sorted from circled area in (A) and 0.5 χ 10 6 of WT or Itk −/− naïve cells (CD45.2 + ) were transferred into lymphopenic Rag −/− , Rag −/− yc −/− , NSG, or sublethally irradiated CD45.2-congenic hosts. (C) Representative plots of WT and Itk −/− CD8 + T cells following 10 days of HP; summary of percentage and number of HP CD8 + T cells of viable lymphocytes in the indicated lymphopenic hosts. Total number of donor CD8 + T cells in the spleen and lymph node of lymphopenic recipients is shown. Data presented as Mean ± SEM; p values generated by non-parametric Mann-Whitney test. N = 4. Data represent results of 3 independent experiments.

Article Snippet: All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target (annotation and clone)” format as follows: eFluor 450-CD122 (IL-2Rβ/IL-15Rα, TM-b1), eFluor 450-H-2Kb (AF6-88.5.5.3), FITC-IL-2 (JES6-5H4), PE-T-bet (eBio4B10), Alexa Fluor 700-CD45.2 (104), PerCP-Cy5.5-CD 127 (IL-7Ra, A7R34), PerCP-eFluor 710-Eomes (Dan11mag), PE-Cy7-IFNγ (XMG1.2), and PE-Cy7-NKG2D (CX5) were purchased from eBioscience (San Diego, CA); V500-CD44 (IM7), FITC-CD45.1 (A20), FITC-CD45.2 (104), FITC-Fas (Jo2), FITC-TCRVβ5 (MR9-4), PE-Annexin V, PE-CD18 (C71/16), PE-IL-4Ra (mIL4R-M1), PE-TCRVa2 (B20.1), PE-TNF-a (MP6-XT22), PE-CF594-CD8a (53-6.7), Cy5-Annexin V, Alexa Fluor 700-CD62L (MEL-14), Alexa Fluor 700-Ki67 (B56) and Allophycocyanin-Cy7-TCRVa2 (B20.1) were purchased from BD Biosciences (San Diego, CA); PE-Texas Red-CD8a (5H10) were purchased from Invitrogen (Carlsbad, CA); FITC-Bcl-2 (10C4), FITC-KLRG1 (2F1), FITC-CD11a (2D7), Allophycocyanin-CD132 (γc, TUGm2), Alexa Fluor 700-CD45.1 (A20), PE-Cy7-CD5 (53-7.3), and PE-Cy7-CD62L (MEL-14) were from Biolegend (San Diego, CA).

Techniques: Expressing, Transgenic Assay, Irradiation, Generated, MANN-WHITNEY

Journal: bioRxiv

Article Title: TCR/ITK signaling via mTOR tunes CD8 + T cell homeostatic proliferation, metabolism, and anti-tumor effector function

doi: 10.1101/359604

Figure Lengend Snippet: (A) Purified congenic naïve WT (CD45.1 + ) and Itk −/− (CD45.1) OTI- Rag −/− CD8 + T cells were mixed at the ratio of 1:1, and a total 120,000 T cells were transferred to Rag −/− lymphopenic recipients, and analyzed 10 and 60 days post transfer in (B-C). N = 4 - 6, from 2 independent experiments. (B) Representative plots and (C) summary of fractions of WT and Itk −/− CD8 + T cells expanded in lymphopenic environment. Data represent Mean ± SEM. p values generated by non-parametric Mann-Whitney test, comparing WT and Itk −/− . (D-F) 1:1 mixture of WT and Itk −/− naïve OTI- Rag −/− CD8 + T cells (total 120,000) were transferred into B2m −/− Rag −/− recipients, and analyzed 10 and 60 days post transfer. N = 3 - 6, from 2 independent experiments. (D) Representative plots and (E) summary of fractions of WT and Itk −/− T cells expanded. p values generated by non-parametric Mann-Whitney test, comparing WT and Itk −/− at each time point. (F) Number of expanded WT and Itk −/− CD8 + T cells. p value generated by two-way ANOVA. (G-I) 1:1 mixed WT and Itk −/− naïve OTI-Rag −/− CD8 + T cells (total 120,000 or 2,000) were transferred into B2m −/− Rag −/− recipients, and analyzed 10 days post transfer. N > 4, combined from independent experiments. p values generated by non-parametric Mann-Whitney test. N = 4 - 5, combined from 2 independent experiments. Data represent Mean ± SEM. (G) Representative plots and (H) summary of fractions of WT and Itk −/− T cells expanded in lymphopenic environment. (I) Number of WT and Itk −/− CD8 + T cells expanded from 120,000 (left y-axis) or 2,000 (right y-axis) initial cells. (J) Fold expansion of 1:1 mixed WT and Itk −/− CD8 + T cells in the same environment: 120,000 initial cells in Rag −/− or B2m −/− Rag −/− recipients, or 2,000 initial cells in B2m −/− Rag −/− recipients. NS = “No Significance”.

Article Snippet: All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target (annotation and clone)” format as follows: eFluor 450-CD122 (IL-2Rβ/IL-15Rα, TM-b1), eFluor 450-H-2Kb (AF6-88.5.5.3), FITC-IL-2 (JES6-5H4), PE-T-bet (eBio4B10), Alexa Fluor 700-CD45.2 (104), PerCP-Cy5.5-CD 127 (IL-7Ra, A7R34), PerCP-eFluor 710-Eomes (Dan11mag), PE-Cy7-IFNγ (XMG1.2), and PE-Cy7-NKG2D (CX5) were purchased from eBioscience (San Diego, CA); V500-CD44 (IM7), FITC-CD45.1 (A20), FITC-CD45.2 (104), FITC-Fas (Jo2), FITC-TCRVβ5 (MR9-4), PE-Annexin V, PE-CD18 (C71/16), PE-IL-4Ra (mIL4R-M1), PE-TCRVa2 (B20.1), PE-TNF-a (MP6-XT22), PE-CF594-CD8a (53-6.7), Cy5-Annexin V, Alexa Fluor 700-CD62L (MEL-14), Alexa Fluor 700-Ki67 (B56) and Allophycocyanin-Cy7-TCRVa2 (B20.1) were purchased from BD Biosciences (San Diego, CA); PE-Texas Red-CD8a (5H10) were purchased from Invitrogen (Carlsbad, CA); FITC-Bcl-2 (10C4), FITC-KLRG1 (2F1), FITC-CD11a (2D7), Allophycocyanin-CD132 (γc, TUGm2), Alexa Fluor 700-CD45.1 (A20), PE-Cy7-CD5 (53-7.3), and PE-Cy7-CD62L (MEL-14) were from Biolegend (San Diego, CA).

Techniques: Purification, Generated, MANN-WHITNEY

Journal: bioRxiv

Article Title: TCR/ITK signaling via mTOR tunes CD8 + T cell homeostatic proliferation, metabolism, and anti-tumor effector function

doi: 10.1101/359604

Figure Lengend Snippet: (A-C) 120,000 naïve WT or Itk −/− OTI- Rag −/− CD8 + T cells are transferred into B2m −/− Rag −/− recipients. N = 3. (A) Representative plots indicating abundance of WT and Itk −/− CD8 + T cells expanded 10 days (blood) or 25 days (spleen and lymph node) post transfer. (B) Number (sum of spleen and lymph node) of WT and Itk −/− CD8 + T cells expanded 25 days post transfer. (C) Representative plots and summary of MFI of IFN-γ and TNF-α expression induced by MHC1/OVA257-264 peptide stimulation of CD8 + T cells expanded 25 days in spleen of B2m −/− Rag −/− hosts. (D-F) EL4 or EG7 (EL4-OVA) lymphoma cells were subcutaneously implanted in the flank of mice (CD45.1 + ) in a contralateral manner, followed by infusion of CD45.2 + WT or Itk −/− HP OTI- Rag −/− CD8 + T cells (previously expanded for 25 days in B2m −/− Rag −/− hosts as in A-C) 2 days later. N = 7 - 9. (D) EL4 and (E) EG7 tumor size in mice that received no cells (control), WT HP or Itk −/− HP CD8 + T cells along the time course post tumor inoculation. (F) EG7 tumor weight on day 18. Data represent Mean ± SEM. p values in (B, D & E) were generated by two-way ANOVA, in (C & F) were generated by Student’s t test. NS = “No Significance”.

Article Snippet: All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target (annotation and clone)” format as follows: eFluor 450-CD122 (IL-2Rβ/IL-15Rα, TM-b1), eFluor 450-H-2Kb (AF6-88.5.5.3), FITC-IL-2 (JES6-5H4), PE-T-bet (eBio4B10), Alexa Fluor 700-CD45.2 (104), PerCP-Cy5.5-CD 127 (IL-7Ra, A7R34), PerCP-eFluor 710-Eomes (Dan11mag), PE-Cy7-IFNγ (XMG1.2), and PE-Cy7-NKG2D (CX5) were purchased from eBioscience (San Diego, CA); V500-CD44 (IM7), FITC-CD45.1 (A20), FITC-CD45.2 (104), FITC-Fas (Jo2), FITC-TCRVβ5 (MR9-4), PE-Annexin V, PE-CD18 (C71/16), PE-IL-4Ra (mIL4R-M1), PE-TCRVa2 (B20.1), PE-TNF-a (MP6-XT22), PE-CF594-CD8a (53-6.7), Cy5-Annexin V, Alexa Fluor 700-CD62L (MEL-14), Alexa Fluor 700-Ki67 (B56) and Allophycocyanin-Cy7-TCRVa2 (B20.1) were purchased from BD Biosciences (San Diego, CA); PE-Texas Red-CD8a (5H10) were purchased from Invitrogen (Carlsbad, CA); FITC-Bcl-2 (10C4), FITC-KLRG1 (2F1), FITC-CD11a (2D7), Allophycocyanin-CD132 (γc, TUGm2), Alexa Fluor 700-CD45.1 (A20), PE-Cy7-CD5 (53-7.3), and PE-Cy7-CD62L (MEL-14) were from Biolegend (San Diego, CA).

Techniques: Expressing, Generated

Journal: bioRxiv

Article Title: TCR/ITK signaling via mTOR tunes CD8 + T cell homeostatic proliferation, metabolism, and anti-tumor effector function

doi: 10.1101/359604

Figure Lengend Snippet: (A&C) 120,000 naïve WT or Itk −/− OTI- Rag −/− CD8 + T cells are transferred into Rag −/− recipients. N = 3–7. (B&D) Congenic naïve WT (CD45.1 + ) and Itk −/− (CD45.1 − ) OTI- Rag −/− CD8 + T cells were mixed at a ratio of 1:1, and a total 120,000 T cells were transferred to Rag −/− recipients. N = 3. (A) Dynamics of expression (Mean Fluorescent Intensity, MFI) of Fas and Bcl-2. (B) Percentage of CD44 hi KI67 + and AnnexinV + cells over CD8 + T cells expanded during lymphopenia-driven HP in the co-transfer model. (C&D) Dynamics of expression (MFI) of LFA subunits CD11a and CD18 in the singly transferred (C) and co-transferred (D) cells. Data represent Mean ± SEM. p values were generated by two-way ANOVA. Data represent Mean ± SEM. NS = “No Significance”.

Article Snippet: All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target (annotation and clone)” format as follows: eFluor 450-CD122 (IL-2Rβ/IL-15Rα, TM-b1), eFluor 450-H-2Kb (AF6-88.5.5.3), FITC-IL-2 (JES6-5H4), PE-T-bet (eBio4B10), Alexa Fluor 700-CD45.2 (104), PerCP-Cy5.5-CD 127 (IL-7Ra, A7R34), PerCP-eFluor 710-Eomes (Dan11mag), PE-Cy7-IFNγ (XMG1.2), and PE-Cy7-NKG2D (CX5) were purchased from eBioscience (San Diego, CA); V500-CD44 (IM7), FITC-CD45.1 (A20), FITC-CD45.2 (104), FITC-Fas (Jo2), FITC-TCRVβ5 (MR9-4), PE-Annexin V, PE-CD18 (C71/16), PE-IL-4Ra (mIL4R-M1), PE-TCRVa2 (B20.1), PE-TNF-a (MP6-XT22), PE-CF594-CD8a (53-6.7), Cy5-Annexin V, Alexa Fluor 700-CD62L (MEL-14), Alexa Fluor 700-Ki67 (B56) and Allophycocyanin-Cy7-TCRVa2 (B20.1) were purchased from BD Biosciences (San Diego, CA); PE-Texas Red-CD8a (5H10) were purchased from Invitrogen (Carlsbad, CA); FITC-Bcl-2 (10C4), FITC-KLRG1 (2F1), FITC-CD11a (2D7), Allophycocyanin-CD132 (γc, TUGm2), Alexa Fluor 700-CD45.1 (A20), PE-Cy7-CD5 (53-7.3), and PE-Cy7-CD62L (MEL-14) were from Biolegend (San Diego, CA).

Techniques: Expressing, Generated

Journal: bioRxiv

Article Title: TCR/ITK signaling via mTOR tunes CD8 + T cell homeostatic proliferation, metabolism, and anti-tumor effector function

doi: 10.1101/359604

Figure Lengend Snippet: TCR activation tunes down CD8 + T cell metabolism and HP via ITK signaling. (A) WT or Itk −/− naïve CD8 + T cells were isolated and immediately analyzed in Seahorse assay media for ECAR and OCR. N = 6. p ≤ *0.05, **0.01, ***<0.001 by two way ANOVA with Tukey post test. Data represent results of more than 3 experiments. (B) WT or Itk −/− naïve CD8 + T cells were cultured in Seahorse assay media with IL-7 overnight, and analyzed for ECAR and OCR. N = 9 pooled from 3 independent experiments. p values by two-way ANOVA. (C) WT or Itk −/− naïve CD8 + T cells were cultured in Seahorse assay media with IL-7 in the presence or absence of αCD3 (0.01 μg/ml) from time 0, and monitored for ECAR and OCR for an overnight. N = 5. (D&E) WT or Itk −/− naïve CD8 + T cells were transferred into Rag −/− recipients on day 0, and recipients received intraperitoneal injection of 0.1 μg/mouse anti-CD3s (or isotype as control) on day 1 and day 3. Spleen and lymph nodes were analyzed on day 10. N = 5. (D) Representative plots of CD8 + T cells in the spleen and lymph node on day. (E) Total numbers of donor CD8 + T cells in the spleen and lymph node on day 10 and fold expansion by normalization to the number in “WT + Iso” group. p values generated by one-way ANOVA with Tukey post test. (F&G) WT (CD45.1) and Itk −/− (CD45.2) naïve CD8 + T cells were mixed in a 9:1 ratio (total each mouse) and transferred into Rag −/− recipients on day 0, and recipients received intraperitoneal injection of 0.1 μg/mouse anti-CD3s (or isotype as control) on day 1 and day 3. Spleen and lymph nodes were analyzed on day 10. (F) Representative plots of CD8 + T cells in the spleen and lymph node on day 10. Cells were pregated on Vα2 + CD8 + and analyzed for CD45.1 to reveal the composition of the final population. (G) Total numbers of donor CD8 + T cells in the spleen and lymph node on day 10 and relative fold expansion by normalization to the number in “WT + Iso” group and to the “9:1” ratio of initial population. p values generated by one-way ANOVA with Tukey post test.

Article Snippet: All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target (annotation and clone)” format as follows: eFluor 450-CD122 (IL-2Rβ/IL-15Rα, TM-b1), eFluor 450-H-2Kb (AF6-88.5.5.3), FITC-IL-2 (JES6-5H4), PE-T-bet (eBio4B10), Alexa Fluor 700-CD45.2 (104), PerCP-Cy5.5-CD 127 (IL-7Ra, A7R34), PerCP-eFluor 710-Eomes (Dan11mag), PE-Cy7-IFNγ (XMG1.2), and PE-Cy7-NKG2D (CX5) were purchased from eBioscience (San Diego, CA); V500-CD44 (IM7), FITC-CD45.1 (A20), FITC-CD45.2 (104), FITC-Fas (Jo2), FITC-TCRVβ5 (MR9-4), PE-Annexin V, PE-CD18 (C71/16), PE-IL-4Ra (mIL4R-M1), PE-TCRVa2 (B20.1), PE-TNF-a (MP6-XT22), PE-CF594-CD8a (53-6.7), Cy5-Annexin V, Alexa Fluor 700-CD62L (MEL-14), Alexa Fluor 700-Ki67 (B56) and Allophycocyanin-Cy7-TCRVa2 (B20.1) were purchased from BD Biosciences (San Diego, CA); PE-Texas Red-CD8a (5H10) were purchased from Invitrogen (Carlsbad, CA); FITC-Bcl-2 (10C4), FITC-KLRG1 (2F1), FITC-CD11a (2D7), Allophycocyanin-CD132 (γc, TUGm2), Alexa Fluor 700-CD45.1 (A20), PE-Cy7-CD5 (53-7.3), and PE-Cy7-CD62L (MEL-14) were from Biolegend (San Diego, CA).

Techniques: Activation Assay, Isolation, Cell Culture, Injection, Generated

Journal: bioRxiv

Article Title: TCR/ITK signaling via mTOR tunes CD8 + T cell homeostatic proliferation, metabolism, and anti-tumor effector function

doi: 10.1101/359604

Figure Lengend Snippet: ITK suppresses CD8 + T cell anti-tumor immunity developed during lymphopenia-induced HP. CD45.1 + WT mice were implanted with EL4 or EG7 (EL4-OVA) lymphoma cells, and received CD45.2 + WT or Itk −/− HP CD8 + T cells (120,000) 2 days later. Tumor size in mice that received no cells (control), WT HP or Itk −/− HP CD8 + T cells (expanded in Rag −/− hosts) along the time course post tumor inoculation. (A) EL4 (N = 7) and (B) EG7 (N = 10) tumor size over time. (C) EG7 tumor weight on day 15. (D-G) CD45.2 + HP CD8 + T cells in mice shown in (B&C) were analyzed on day 15. (D) Inverse correlation between the number of Itk −/− HP CD8 + T cells in the tumor site and tumor size. R 2 implicates the degree of correlation and P reflects the likelihood of incorrect prediction. (E) Numbers of CD45.2 + HP CD8 + T cells in draining lymph node (LN) and tumor site. (F) Expression of PD-1, CTLA-4, and CD107a (MFI) by HP CD8 + T cells in draining lymph node and tumor site. (G) CD45.2 + HP CD8 + T cells from draining lymph nodes of tumor recipients were stimulated with P/I or OVA 257-264 and IFN-γ and TNF-α production determined by FACS. Data represent Mean ± SEM. p values in (A&B) generated by two-way ANOVA, in (C, E, F & G) generated by non-parametric Mann-Whitney test. NS = “No Significance”.

Article Snippet: All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target (annotation and clone)” format as follows: eFluor 450-CD122 (IL-2Rβ/IL-15Rα, TM-b1), eFluor 450-H-2Kb (AF6-88.5.5.3), FITC-IL-2 (JES6-5H4), PE-T-bet (eBio4B10), Alexa Fluor 700-CD45.2 (104), PerCP-Cy5.5-CD 127 (IL-7Ra, A7R34), PerCP-eFluor 710-Eomes (Dan11mag), PE-Cy7-IFNγ (XMG1.2), and PE-Cy7-NKG2D (CX5) were purchased from eBioscience (San Diego, CA); V500-CD44 (IM7), FITC-CD45.1 (A20), FITC-CD45.2 (104), FITC-Fas (Jo2), FITC-TCRVβ5 (MR9-4), PE-Annexin V, PE-CD18 (C71/16), PE-IL-4Ra (mIL4R-M1), PE-TCRVa2 (B20.1), PE-TNF-a (MP6-XT22), PE-CF594-CD8a (53-6.7), Cy5-Annexin V, Alexa Fluor 700-CD62L (MEL-14), Alexa Fluor 700-Ki67 (B56) and Allophycocyanin-Cy7-TCRVa2 (B20.1) were purchased from BD Biosciences (San Diego, CA); PE-Texas Red-CD8a (5H10) were purchased from Invitrogen (Carlsbad, CA); FITC-Bcl-2 (10C4), FITC-KLRG1 (2F1), FITC-CD11a (2D7), Allophycocyanin-CD132 (γc, TUGm2), Alexa Fluor 700-CD45.1 (A20), PE-Cy7-CD5 (53-7.3), and PE-Cy7-CD62L (MEL-14) were from Biolegend (San Diego, CA).

Techniques: Expressing, Generated, MANN-WHITNEY

Journal: bioRxiv

Article Title: TCR/ITK signaling via mTOR tunes CD8 + T cell homeostatic proliferation, metabolism, and anti-tumor effector function

doi: 10.1101/359604

Figure Lengend Snippet: Naïve CD45.1 + WT and CD45.2 + Itk −/− OTI- Rag −/− CD8 + T cells (initial total number ≈ 0.1 × 10 6 for each recipient) were expanded concurrently in Rag −/− or B2m −/− Rag −/− hosts for 10 and 60 days, followed by stimulation with OVA257–264 peptide in vitro. CD45.1 + WT and CD45.2 + Itk −/− HP CD8 + T cells in the spleen of lymphopenic hosts were analyzed. (A) Representative plots of IFN-γ expression induced by OVA257–264 stimulation. Mock-stimulated (PBS/BFA) cells were used as background controls. Average percentage of IFN-γ producing cells shown on plot: WT in grey and Itk −/− in black. (B) Percentage and MFI of IFN-γ producing HP cells. Fold changes were derived by dividing the levels on day 60 by those on day 10, in WT or Itk −/− HP cells respectively. p values were generated by Student’s t test, comparing percentages at the same time point or fold changes connected. (C) Dynamic expression (MFI) of CD5, CD8a and TCR (Vα2) in WT or Itk −/− CD8 + T cells during lymphopenia-induced HP. p values were generated by two-way ANOVA. N = 2 - 7. Data represent Mean ± SEM. NS = “No Significance”.

Article Snippet: All fluorochrome-conjugated antibodies used are listed in “fluorochrome-target (annotation and clone)” format as follows: eFluor 450-CD122 (IL-2Rβ/IL-15Rα, TM-b1), eFluor 450-H-2Kb (AF6-88.5.5.3), FITC-IL-2 (JES6-5H4), PE-T-bet (eBio4B10), Alexa Fluor 700-CD45.2 (104), PerCP-Cy5.5-CD 127 (IL-7Ra, A7R34), PerCP-eFluor 710-Eomes (Dan11mag), PE-Cy7-IFNγ (XMG1.2), and PE-Cy7-NKG2D (CX5) were purchased from eBioscience (San Diego, CA); V500-CD44 (IM7), FITC-CD45.1 (A20), FITC-CD45.2 (104), FITC-Fas (Jo2), FITC-TCRVβ5 (MR9-4), PE-Annexin V, PE-CD18 (C71/16), PE-IL-4Ra (mIL4R-M1), PE-TCRVa2 (B20.1), PE-TNF-a (MP6-XT22), PE-CF594-CD8a (53-6.7), Cy5-Annexin V, Alexa Fluor 700-CD62L (MEL-14), Alexa Fluor 700-Ki67 (B56) and Allophycocyanin-Cy7-TCRVa2 (B20.1) were purchased from BD Biosciences (San Diego, CA); PE-Texas Red-CD8a (5H10) were purchased from Invitrogen (Carlsbad, CA); FITC-Bcl-2 (10C4), FITC-KLRG1 (2F1), FITC-CD11a (2D7), Allophycocyanin-CD132 (γc, TUGm2), Alexa Fluor 700-CD45.1 (A20), PE-Cy7-CD5 (53-7.3), and PE-Cy7-CD62L (MEL-14) were from Biolegend (San Diego, CA).

Techniques: In Vitro, Expressing, Derivative Assay, Generated

Journal: PLoS ONE

Article Title: CXCR4 Is Dispensable for T Cell Egress from Chronically Inflamed Skin via the Afferent Lymph

doi: 10.1371/journal.pone.0095626

Figure Lengend Snippet: ( A–C ) Adoptive transfer exit experiments with fetal liver chimeric mice as a source of donor splenocytes. As depicted in the experimental scheme ( A ), fetal liver chimeras were generated by reconstituting lethally irradiated CD45.1 + wildtype recipient mice with fetal liver cells from (CD45.2 + ) Cxcr4 −/− Ccr 7 −/− or (CD45.2 + ) Ccr7 −/− or (CD45.1 + ) wildtype donors. Splenocytes from the fetal liver chimeras were labeled with eFlour670 and CFSE, so that the combination of congenic maker and fluorescent label uniquely marked cells of each genotype. Labeled splenocytes of all three genotypes were mixed and co-injected into chronically inflamed footpads. ( B ) Analysis of migrated lymphocyte subsets recovered in the draining popliteal lymph nodes 12 h post cell transfer. Data is presented as the ratio of migrated to input cells for Ccr7 −/− or Cxcr4 −/− Ccr 7 −/− relative to wildtype cells of each subset. Connected lines represent individually analyzed recipient mice. Horizontal, non-connecting lines indicate the mean of each group. ( C ) Flow cytometric analysis of memory (CD45RB l °) versus naïve (CD45RB hi ) phenotype CD4 and CD8 T cells in injected and migrated populations for each genotype. Data combined from 2 experiments analyzing 5–10 recipient mice per experiment ( B ) or the cell phenotype of one out two experiment with similar results ( C ) is shown. WT, wildtype.

Article Snippet: The following rat anti-mouse mAbs (all from Ebioscience) were used: CD4 (clone RM4–5), CD8 (clone 53–6.7), CD45RB (clone 16A), CD45.1 (clone A20), and CD45.2 (clone104).

Techniques: Adoptive Transfer Assay, Generated, Irradiation, Labeling, Injection

Journal: The Journal of Experimental Medicine

Article Title: Hierarchy of Notch–Delta interactions promoting T cell lineage commitment and maturation

doi: 10.1084/jem.20061442

Figure Lengend Snippet: N2 signaling is sufficient to induce T lineage commitment in vitro but not in vivo. (A) Mixed BM chimeric mice were analyzed 8 wk after reconstitution with a 1:2 mixture of WT (CD45.1 + ) and Ctrl ( N1 lox/lox ), N1 −/− , N2 −/− , or N1N2 −/− (CD45.2 + ) BM-derived populations. Representative FACS analysis of thymocytes stained with anti-CD117, -CD44, and -CD25 antibodies after gating on donor (CD45.2 + )-derived lineage-negative cells (top). Representative FACS analysis of thymocytes stained with anti-B220 and -CD19 antibodies after gating on donor (CD45.2 + )-derived lineage-negative cells (bottom). Representative FACS profiles are derived from experiments in which five mice of each genotype were analyzed. (B) BM KLS cells were sorted from Ctrl , N1 −/− , N2 −/− , and N1N2 −/− mice and cultured on OP9-DL1 cells for 10 d (top) and 18 d (bottom). Cells from these cultures were analyzed for the expression of CD44 and CD25 (top) and for the presence of B220 + CD19 + B cells (bottom). Representative FACS profiles are derived from four individual experiments. (C) Deletion PCR analysis for the N1 gene was performed on genomic DNA from sorted CD25 − (corresponding to DN1) and CD25 + (corresponding to DN2/DN3) cells derived from N1 −/− and Ctrl animals cultured for 10 d on OP9-DL1. (D) Semiquantitative RT-PCR for the expression of N1 , N2 , and tubulin was performed on sorted BM KLS cells. Three serial dilutions (threefold) of template RNA are shown for the indicated genes.

Article Snippet: The following monoclonal antibody conjugates were purchased from eBioscience: CD117 (2B8)-PE and -PE-Cy5.5; Sca-1 (D7)-PE and -APC; CD19 (MB-19.1)-PE and (6D5)-PE-Cy5.5; B220 (RA3.6B2)-PE-Cy5.5 and –Alexa Fluor 647; CD44 (IM781)-PE-Cy5.5; CD25 (PC61)-APC; CD4 (L3T4)-PE-Cy5.5; CD45.2 (104)-PE-Cy5.5; TCRβ (H57)-PE and -APC; CD161 (PK136)-FITC; CD90.1 (HIS15)-PE; and CD90.2 (30H12)-PE.

Techniques: In Vitro, In Vivo, Derivative Assay, Staining, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Hierarchy of Notch–Delta interactions promoting T cell lineage commitment and maturation

doi: 10.1084/jem.20061442

Figure Lengend Snippet: N2 is sufficient to specify T lineage progenitors in the spleen after BM transplantation. CD45.2 + Ctrl , N1 −/− , N2 −/− , or N1N2 −/− BM cells were injected into lethally irradiated CD45.1 + hosts. The spleens of host mice were analyzed 12 d after BM transplantation. Representative flow cytometric analyses of donor-derived lineage-negative cells for the expression of CD44 and Thy1.2, and Thy1.2 and CD25, respectively, are shown. Data are representative of four independent experiments.

Article Snippet: The following monoclonal antibody conjugates were purchased from eBioscience: CD117 (2B8)-PE and -PE-Cy5.5; Sca-1 (D7)-PE and -APC; CD19 (MB-19.1)-PE and (6D5)-PE-Cy5.5; B220 (RA3.6B2)-PE-Cy5.5 and –Alexa Fluor 647; CD44 (IM781)-PE-Cy5.5; CD25 (PC61)-APC; CD4 (L3T4)-PE-Cy5.5; CD45.2 (104)-PE-Cy5.5; TCRβ (H57)-PE and -APC; CD161 (PK136)-FITC; CD90.1 (HIS15)-PE; and CD90.2 (30H12)-PE.

Techniques: Transplantation Assay, Injection, Irradiation, Derivative Assay, Expressing

Journal: Environmental Health Perspectives

Article Title: Oral Exposure to Polystyrene Microplastics of Mice on a Normal or High-Fat Diet and Intestinal and Metabolic Outcomes

doi: 10.1289/EHP11072

Figure Lengend Snippet: Histological evaluation of jejunum and immune cells involved in innate immunity in LPL of small intestine. (A) Representative images of HE- and PAS-stained, GFP-positive, and Muc2-immunostained jejunum sections. Jejunum tissue was collected at 12 wk of age. In the GFP fluorescence image, MPs are enlarged and indicated by arrows. The scale bars show 100 μ m ( 50 μ m for Muc2 image). (B) Villus height ( n = 10 ). (C) Villus width ( n = 10 ). (D) Crypt depth ( n = 10 ). (E) Total goblet cells/area ( mm 2 of jejunum) ( n = 10 ). (F) Mucus layer thickness ( n = 10 ). (G) GFP-positive area ( n = 10 ). Ratio of (H) ILC1s to CD45-positive cells, (I) T-bet positive ILC3s to CD45-positive cells, (J) M1 macrophages to M2 macrophages in the small intestine, and (K) ILC3s to CD45-positive cells ( n = 10 in each case). Data are presented as mean ± SD values. Data were analyzed using one-way ANOVA with Holm-Šídák’s multiple comparisons test. Summary data can be found in Table S2. * p < 0.05 , ** p < 0.01 , *** p < 0.001 , and **** p < 0.0001 . Note: ANOVA, analysis of variance; GFP, green fluorescent protein; H&E, hematoxylin and eosin; HFD, high-fat diet; ILCs, innate lymphoid cells; MPs, microplastics; ND, normal diet; PAS, periodic acid Schiff; SD, standard deviation.

Article Snippet: We used the following antibodies for gating of M1 and M2 macrophages: FITC-CD206 (MA516870; clone: MR5D3, 1/50, eBioscience, Inc.), PE-F4/80 (12,480,182; clone: BM8, 1/50, eBioscience, Inc.), APC- CD45.2 (17,045,482; clone: 104, 1/50; eBioscience, Inc.), PE-Cy7-CD11c (25,011,482; clone: N418, 1/50, eBioscience, Inc.), and APC-Cy7-CD11b (47,011,282; clone: M1/70, 1/50; eBioscience, Inc.) (Figure S2).

Techniques: Staining, Fluorescence, Standard Deviation

Journal: JCI Insight

Article Title: Siponimod therapy implicates Th17 cells in a preclinical model of subpial cortical injury

doi: 10.1172/jci.insight.132522

Figure Lengend Snippet: (A–F) Single-cell suspensions from the brains of BAF312- versus control-treated mice at day 11 after adoptive transfer were stained for CD4 as well as CD45.1 versus CD45.2 alleles identifying endogenous (CD45.1 host-derived) versus transferred (CD45.2 donor-derived) lymphocytes. (A and B) Total CD4+ T cells were assessed, and (C–F) cytokine production from CD4+ T cells was measured using intracellular staining for IFN-γ, IL-17, and GM-CSF as well as for both IFN-γ and IL-17 or both GM-CSF and IL-17. Values are expressed either as a frequency of total live cells (A, C, E) or as an absolute number of brain-resident cells (B, D, F). Two experiments were combined for analysis; n = 8–9 mice per group. Values are shown as mean ± SD, and statistical significance was determined by Mann-Whitney U, with *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: This was immediately followed by staining with anti–CD4-FITC (eBioscience, Thermo Fisher Scientific), anti–CD8-BV711 (eBioscience, Thermo Fisher Scientific), anti–CD3-APC (eBioscience, Thermo Fisher Scientific), anti–B220-BV605 (BioLegend), anti–CD19-PE (eBioscience, Thermo Fisher Scientific), anti–CD45.1-APCeF780 (eBioscience, Thermo Fisher Scientific), and anti–CD45.2-eF450 (eBioscience, Thermo Fisher Scientific) in PBS with 2% FBS and 1 mg/mL Fc block.

Techniques: Adoptive Transfer Assay, Staining, Derivative Assay, MANN-WHITNEY