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: The Journal of Experimental Medicine

Article Title: An immunoglobulin Cκ-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system

doi: 10.1084/jem.20041854

Figure Lengend Snippet: Flow cytometry analysis of tolerance induction in radiation chimeras using κ-macroself transgenic hosts. Mice were analyzed at 6 wk post reconstitution. (A and B) Analysis of bone marrow lymphocytes using anti-L chain and B220 antibodies. Bone marrow donors are indicated to the left of the arrows, the recipient mouse genotypes are shown just below. Newly formed lymphocytes carrying Ig-κ or -λ, were identified as falling in the lower analysis boxes, as indicated. (A) B6 (wt) or RAG +/− bone marrow was used to reconstitute lethally irradiated CD45.1 + /κ-macroself transgenic #2 (Tg #2) or littermate recipients. (B) Comparison of chimeras generated with bone marrow from Bcl-2 transgenic or littermate (wt) donors, using as irradiated recipients either transgenic #2 or littermate. (C) Analysis of spleen cells from the indicated radiation chimeras. Recipient mouse genotype is shown to the right of arrows above dot plots. Cells were stained with B220 and anti-κ antibodies (top) or anti-κ and anti-λ antibodies (bottom).

Article Snippet: Cells were stained in staining buffer containing appropriately diluted combinations of the following monoclonal antibodies: biotin-coupled mouse anti–rat IgG1 (BD Biosciences) developed with streptavidin-PE; PE and biotin rat anti–mouse Ig κ (187.1; BD Biosciences); biotin–anti-Ig λ 1-3 (BD Biosciences) followed by PE- or allophycocyanin-streptavidin; PerCP- or FITC-coupled anti-CD45R/B220 (RA3-6B2; BD Biosciences); Cy5-coupled anti-IgM (331.12), PE-coupled anti-CD45.1 (eBioscience); FITC-coupled anti-CD45.2 (eBioscience).

Techniques: Flow Cytometry, Transgenic Assay, Irradiation, Generated, Staining

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: 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