14-5825-80

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CD4+ T cells were sorted from mouse spleen, activated with Anti-Mouse CD3 and Anti-Mouse CD28, followed by culture in Th1-polarizing conditions, and re-activation with PMA and Ionomycin. Lysates from control (left) or PMA and Ionomycin-re-activated (right) cells were probed with Anti-Human/Mouse T-bet Purified at 2 µg/mL, and revealed with Anti-Mouse IgG HRP.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CD4+ T cells were sorted from mouse spleen, activated with Anti-Mouse CD3 and Anti-Mouse CD28, followed by culture in Th1-polarizing conditions, and re-activation with PMA and Ionomycin. Lysates from control (left) or PMA and Ionomycin-re-activated (right) cells were probed with Anti-Human/Mouse T-bet Purified at 2 µg/mL, and revealed with Anti-Mouse IgG HRP.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CD4+ T cells were sorted from mouse spleen, activated with Anti-Mouse CD3 and Anti-Mouse CD28, followed by culture in Th1-polarizing conditions, and re-activation with PMA and Ionomycin. Lysates from control (left) or PMA and Ionomycin-re-activated (right) cells were probed with Anti-Human/Mouse T-bet Purified at 2 µg/mL, and revealed with Anti-Mouse IgG HRP.

Supportive validation

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Experimental details: Knockdown of T-bet was achieved by transfecting NK-92 with T-bet specific siRNAs (Silencer® select Product # s26889, s223839). Western blot analysis (Fig. a) was performed using modified whole cell extracts (1% SDS) from the T-bet knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with T-bet Monoclonal Antibody (Product # 14-5825-82, 2ug/ml) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to T-bet.

Supportive validation

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Experimental details: Western blot was performed using Anti-T-bet Monoclonal Antibody (Product # 14-5825-82) and a 58kDa band corresponding to T-bet was observed across cell lines tested and the protein was not expressed in K-562. Modified whole cell extracts (1% SDS) (30 µg lysate) of NK-92 (Lane 1) and K-562 (Lane 2) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (2ug/ml) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: Figure 2 1 wk ABX gvg-treated APP SWE /PS1 DeltaE9 mice display altered peripheral and brain inflammatory profiles. ( A ) Representative density dot plots of T-bet and Foxp3 intracellular expression in TCRbeta + CD4 + T cell populations isolated from MLN, blood and brain tissue of vehicle and 1 wk ABX gvg-treated APP SWE /PS1 DeltaE9 mice analysed by flow cytometry. Quantified percentages of ( B ) Foxp3 + and ( C ) T-bet + CD4 + T cells, representative of a T-reg and Th1 T cell phenotype respectively, are expressed relative to total live CD4 + T cell counts ( n = 5-6, *p < 0.05, **p < 0.01, un-paired two-tailed Student's t -test). ( D ) Immunoblot-based array of inflammatory mediators present in the serum of vehicle and 1 wk ABX gvg-treated APP SWE /PS1 DeltaE9 mice ( n = 10 pooled sera). ( E ) Immunoblot-based array of inflammatory mediators present in the CSF of vehicle and 1 wk ABX gvg-treated APP SWE /PS1 DeltaE9 mice ( n = 10 pooled CSF). ( F ) Heat map analysis of inflammatory mediator fold change expression in 1 wk ABX gvg-treated APP SWE /PS1 DeltaE9 mice relative to control. Data are displayed as log e (mean) or mean +- SEM. See Supp. Figs 2 , 4 , 5 , 6 , and statistical Table 2 for additional information.

Supportive validation

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Experimental details: Figure 4 The mTORC1 regulated CTL proteome (a) Immunoblot analysis of mTORC1/2 substrates in P14 TCR transgenic CTLs cultured with IL-2/IL-12 +- 48 h treatment with either rapamycin or KU-0063794. (b) Protein synthesis was examined by monitoring 3 H-Met incorporation into nascent proteins in CTLs cultured in IL-2/IL-12 and treated with rapamycin for the indicated time. (c) Cellular protein content of CTLs +- 48 h rapamycin. (d, e,) Volcano plots showing fold changes in proteins vs. log-transformed P -values from mass spectrometry analysis of CTLs +- 48 h rapamycin. (d) Total proteins. Known rapamycin sensitive proteins perforin and L-selectin are highlighted. (e) CTL effector molecules. (f) IFN-gamma secretion by CTLs +- 48 h rapamycin measured by ELISA. (h) Immunoblot analysis of T-bet in CTLs +- 48 h rapamycin. (h, i) Validation of up-regulated proteins: (h) ELISA of shed CD62L in cell supernatants prepared from CTLs +- 48 h rapamycin. (i) Immunoblot analysis of IRS2 in CTLs +- 48 h rapamycin. (a, g, i): representive immunoblots of at least three biological replicates. (b, c, f, h): individual data points and means are shown. P -values shown determined by (b): one-way ANOVA (Holm-Sidak) vs. DMSO as control on non-normalized data; (c, f, h): two-tailed Student's t-test. Data based on three (b, f, h) or four (c) biological replicates. * P

Supportive validation

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Experimental details: Fig. 2 Absence of CD160 does not affect the development of NKT cells. a Lymphocytes from blood, thymus, liver, and spleen in WT and CD160 -/- mice were stained with anti-TCRbeta FITC and PBS57-CD1d tetramer PE and analyzed by flow cytometry. Results are representative of three independent experiments. The absolute number of TCRbeta + PBS57-CD1d Tetramer + NKT cells in the thymus, liver, and spleen was calculated based on the percentage of each population shown in ( n = 3 per group). b Flow cytometry analysis of CD44 and NK1.1 expression among total NKT cells from the thymus of WT and CD160 -/- mice. Results are representative of three independent experiments. The percentages and numbers of thymic NKT cells at stage 0 (NK1.1 - , CD44 - , CD24 + ), stage 1 (NK1.1 - , CD44 - , CD24 - ), stage 2 (NK1.1 - , CD44 + ), and stage 3 (NK1.1 + , CD44 + ), based on flow cytometry analysis. Results are representative of three independent experiments. ( n = 5-6 per group). c Thymic NKT cells were gated on PLZF, T-bet and RORgammat and the percentage and absolute numbers of NKT1, NKT2, and NKT17 were calculated ( n = 5-6 per group). All data are presented as mean +- S.E.M. * P < 0.05, ** P < 0.01, *** P < 0.001 with an unpaired two-tailed T -test

Supportive validation

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Experimental details: Fig 5 Lentiviral-mmu-miR-135a treatment influences Th cell polarization. The expression of T-bet and GATA-3 protein in CD4 + T cells was measured in the spleens of normal (control), AR (AR-induced), positive (AR-induced, treated with lentiviral-mmu-miR-135a), and negative (AR-induced, treated with empty lentivirus) mice using flow cytometry. (A) Representative dot plots from each experimental group. The percentages of CD4 + T-bet + T cells (B) and CD4 + GATA-3 + T cells (C) were also calculated. Data are presented as the mean +- SEM. *P

Supportive validation

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Experimental details: Figure 7. Melanoma Patients Demonstrated an Overall Increase of PS Expression on Immune Subsets in the Blood 4-7 Days after RT Peripheral blood was collected from 7 melanoma patients before and 4-7 days after receiving tumor-directed RT. Freshly isolated PBMCs from each patient were stained for PS expression using annexin V on the day the blood was collected, as described in STAR Methods . (A) Histogram plots of annexin V staining of viable immune cell subsets in PBMCs from a single patient (Pt. 1) pretreatment (Tx) and post-RT. (B) Representative plots gated on live CD3+ CD8+ T cells of annexin V versus caspase-3/7 activity of PBMCs from a single patient pre-RT and 4 days post-RT. FMO, control. (C) Top: individual values for each patient. Bottom: average percentage +- SEM of annexin V+ immune cell subsets pre-RT and post-RT. *p < 0.05, ***p < 0.005. (D) Model summarizing the effects of targeting PS with RT and anti-PD-1 on immune cell activation.

Supportive validation

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Experimental details: Figure 4 Treg population in MLN is influenced by the dietary intervention. Cells from MLNs were isolated from mice (n = 7 per group) receiving either placebo or scGOS/lcFOS/pAOS and were labeled with CD4/Foxp3 in combination with CXCR3, T-bet, for flowcytometric analysis (A). Lines represent mean percentages of Tregs in total (CD4 + Foxp3 + T cells) (B), and sub-populations of Tregs including % of CXCR3 + /T-bet + Tregs (C) In addition, individual measurements are indicated through separate dots. Data presented is representative for 3 individual experiments. Statistically significant differences between the groups are indicated in the graphs.

Supportive validation

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Experimental details: Figure 5 Cellular localization of 3-NT in the OB of aged mice by double-labeling immunohistochemistry with two neuronal markers, anti-NeuN (A-H) and anti-TBX21 (L-N). A-D: Glomerular layer. A: Small and large 3-NT-labeled puncta (red) are seen within the glomeruli (circles) and in the periglomerular region. B: Numerous periglomerular cells localized in the periglomerular region are labeled with anti-NeuN (green), a neuronal cell marker. C: In this merged image of A and B, few 3-NT labeled puncta are localized in NeuN-labeled periglomerular cells. D: A transmission light micrograph overlaid with C. E-H: Mitral cell layer (MCL) and 2 adjacent regions of the external plexiform layer (EPL) and the inner plexiform layer (IPL). E: Large 3-NT-labeled puncta are clearly seen on the border of the MCL and EPL. F: Many NeuN-labeled granule cells located in the MCL are observed. G: In this merged image of E and F, large 3-NT-labeled puncta are not localized to NeuN-labeled granule cells. H: In a transmission light micrograph overlaid with G, the large 3-NT-labeled puncta are exclusively localized externally to putative NeuN-negative mitral cells (asterisks). The rectangular area is magnified in I-K. I-K: A mitral cell. I: In a transmission light micrograph overlaid with red immunofluorescence, the 3-NT-labeled puncta with triangular shape is clearly seen in the apical portion of a putative mitral cell. J: In the confocal image using the red channel for 3-NT immunoreactivity, the labeled

Supportive validation

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Experimental details: Figure 4 Phenotype of artARENA-induced CTLs and their dependence on IL-33-ST2 alarmin signaling (A-F) We immunized BALB/c mice with artPICV-P1A and artLCMV-P1A in homologous or heterologous prime-boost vaccination i.v. on day 0 and day 27. On day 34, we analyzed P1A-Tet-binding and CD62L expression by splenic CD8 + T cells (A; gated on CD8 + B220 - lymphocytes). Unimmunized control mice are shown for comparison in (A) only. Numbers in (A) indicate the percentage of cells in the respective quadrant. Total P1A-Tet + CTLs (B), P1A-specific effector/effector memory CTLs (CD62L lo ; C), and P1A-specific central memory CTLs (CD62L hi ; D) were enumerated in the spleen on day 34. In both subsets of P1A-specific CTLs, CD62L hi and CD62L lo , we determined the surface expression of KLRG1, CX3CR1, CD27, CD43, and CD127 as well as the master transcription factors Tcf-1, Tbet, and Eomes (E). Total numbers of marker-expressing P1A-specific CTLs were enumerated in (F). (A) shows representative FACS plots from individual mice. Symbols in (B)-(D) and (F) represent individual mice, and bars in (B)-(D) and (F) indicate the mean +- SD. Numbers in (A) and (E) indicate the percentage of gated cells (mean +- SD) or the mean fluorescence intensity (MFI +- SD). Means were calculated from six mice per immunization group (A-F) or from three unimmunized controls (A). N = 2. **p < 0.01 by unpaired two-tailed Student''s t test. (G and H) We immunized ST2 -/- and WT mice with artLCMV-E7E6, artPICV-E7E6 or

Supportive validation

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Experimental details: Figure 1. Patients with Parkinson's disease (PD) and mice with experimental PD exhibit decreased regulatory T (Treg) and increased T helper 1 (Th1) cell numbers in the blood. Treg cells were defined as CD3+CD4+CD25+FoxP3+ cells, whereas Th1 cells were identified as CD3+CD4+T-bet+ cells. (a) Representative plots of Treg and Th1 cells in patients with PD and healthy volunteers (HVs). (b) Representative plots of Treg and Th1 cells in control mice (Saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. (c) Patients with PD exhibited lower Treg cell levels and greater Th1 cell levels in the blood than HVs. n = 20. ****, p < 0.0001 by a two-tailed Student's t tests. (d) MPTP-induced experimental PD mice had fewer Treg and more Th1 cells in the circulation than saline-treated mice. n = 9/group in each experiment performed in triplicate. ***, p < 0.001; ****, p < 0.0001, according to Student's t test.

Supportive validation

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Experimental details: Figure 1 Altered distribution of Th1, Th2, and Th17 in PBMCs of patients. (A) PBMCs were stained intracellularly with T-bet, GATA-3, and ROR-gammat mAbs after surface staining of CD4 mAb. According to CD4 staining and SSC, CD4 + T cells were gated. The parameters shown in quadrants of the representative graphs are mean frequency of GATA-3 for each group. (B-D) The average percentages of CD4 + T-bet + Th1, CD4 + GATA-3 + Th2, CD4 + ROR-gammat + Th17 are compared between HC (n = 24) and whole patients (n = 50) as well as subgroups of patients (SD, n = 24; LD, n = 26; FF, n = 33; LF, n = 17). Error bars represent mean+-SD. ** P < 0.01, * P < 0.05, and NS P >= 0.05.

Supportive validation

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Experimental details: Fig. 3 ADAM12 is essential for T-bet expression in T cells and IFNgamma production. a ADAM12 +/+ and ADAM12 -/- T cells were treated with plate-bound anti-CD3 (5 mug/ml) and anti-ADAM12 (10 mug/ml) for 3 days. Cell-culture supernatants were collected for ELISA. MOG 35-55 -specific T cells treated with MOG 35-55 (50 mug/ml), IL-12 (20 ng/ml), anti-IL-4 (10 mug/ml), and siRNAs (siControl or siADAM12) for 72 h. b FACS dot plots of the gating strategy and T-bet staining in CD4 + T cells. c Quantification of FACS results from b . d IFNgamma production in Th1 cell cultures by ELISA. Graphs in c, d are mean +- SEM from three independent experiments. *** P < 0.001 by Student's t test. Th17 cells polarized in vitro. e , f Representative FACS plots of gating strategy and T-bet- and RORgammat-stained CD4 + T cells. g Quantification of FACS results from a representative experiment--i.e., from two independent experiments. Graphs are mean +- SEM, N = 3. h IFNgamma and IL-17 production in Th17 cell cultures, by ELISA from three independent experiments. ** P < 0.01 by two-way ANOVA with post-Tukey's multiple comparisons test

Supportive validation

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Experimental details: Figure 13 Pa MR1T cell antigen-specific proliferation assay (A) Representative FACS plots showing frequencies of Pa MR1T cells (defined as CD3 + hMR1-5-OP-RU + ) and CTV dilution on day 0 and day 5 of culture with 5-OP-RU from a single Pa donor. (B) Representative FACS plots showing the expression levels of different transcription factors (PLZF, T-bet, Eomes, and RORgammat) and Perforin against CTV at the two different timepoints. Figure adapted and reprinted with permission from ().

Supportive validation

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Experimental details: Figure 1. Exhausted CD8 T cells have a high ratio of nuclear Eomes to T-bet that correlates with PD-1 expression during LCMV infection ImageStream analysis was performed on CD8 + T cells from Armstrong-immune (T MEM , blue)- or clone 13 (T EX , red)-infected mice at day 30 p.i. (A) Representative cell images acquired in IDEAS software from an Armstrong-immune mouse (T MEM , left) or chronic clone 13 mouse (T EX, right) are shown. Splenocytes were permeabilized and stained with T-bet (yellow). The location of the nucleus is indicated by DAPI (cyan). (B) Representative ImageStream flow plots displaying T-bet localization in T MEM s or T EX s are shown (left). Bar graphs display the frequency and median fluorescence intensity (MFI) of nuclear T-bet in LCMV-specific H-2D b gp276 + CD8 + T cells (right). (C) Representative cell images acquired in IDEAS software from an Armstrong-immune mouse (T MEM , left) or chronic clone 13 mouse (T EX , right) are shown. Splenocytes were permeabilized and stained with Eomes (magenta). The location of the nucleus is indicated by DAPI (cyan). (D) Representative ImageStream flow plots displaying Eomes localization are shown (left). Bar graphs show the frequency and MFI of nuclear Eomes in LCMV-specific H-2D b gp276 + CD8 + T cells (right). (E) The ratio of the MFI of nuclear Eomes:T-bet in LCMV-specific H-2D b gp276 + T-bet + Eomes + CD8 T cells is shown. (F) A correlation plot displaying the ratio of the MFI of nuclear Eomes:T-bet versus MFI of P

Supportive validation

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Experimental details: Fig. 4 MAIT cells were polarized to functional MAIT-1 phenotype upon F. tularensis infection. A Representative flow cytometry plots showing intranuclear staining for T-bet (representing Th1) and RORgammat (Th17) in gated MAIT cells from the liver, lungs, spleen, kidneys, LPL, IEL, and blood of naive and infected mice on 6 and 40+ dpi (41 and 68 dpi in two experiments) with 10 4 CFU F. tularensis LVS i.v. Numbers in quadrants represent cell percentage. It is noteworthy that IEL from naive mice yielded insufficient numbers of MAIT cells for accurate assessment of transcription factor expression and, thus, were omitted from our analysis. B Percentage of MAIT cells expressing combinations of T-bet and RORgammat from the same mice in A . Pooled data from two independent experiments (mean +- SEM, n = 3-6 mice per group, as indicated). One-way ANOVA with Tukey's multiple comparisons test was performed on MAIT-1% in each organ (except for IEL) between time points as indicated; p -values are indicated, nd; not determined. Source data are provided as a Source Data file.

Supportive validation

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Experimental details: Fig. 7 MAIT cell-mediated protection in immune-compromised mice requires IFNgamma, TNF, and GM-CSF. A MAIT cell percentage of alphabeta-T cells in the liver and representative FACS plot showing intranuclear staining for T-bet and RORgammat of MAIT cells from donor C57BL/6 mice vaccinated with CpG and 5-OP-RU i.v. for 7 days, prior cell sorting for adoptive cell transfer. Pooled data from 7 (nil) or 11 (vaccinated) mice from 3 independent experiments (mean +- SEM). Unpaired t -test (two-tailed). P < 0.0001. B Schematic of protocol for MAIT cell adoptive transfer and F. tularensis LVS challenge: 10 5 liver MAIT cells from C57BL/6 (WT, shown in A ), Ifngamma -/- , Tnf -/- , Gm-csf -/- , or Il-17 -/- mice vaccinated with CpG (10 nmol) and 5-OP-RU (2 nmol) i.v. for 7 days were sorted by flow cytometry and transferred i.v. into Rag2 -/- gammaC -/- mice. The mice were treated with anti-CD4 and anti-CD8 mAb injection (i.p., 0.1 mg each) at days 1 and 3 post MAIT cell transfer, to deplete contaminating conventional T cells. After 2 weeks, mice were infected with an otherwise lethal dose (20 CFU) of F. tularensis LVS i.v. C Survival of untreated Rag2 -/- gammaC -/- mice or Rag2 -/- gammaC -/- mice following transfer of MAIT cells from WT, Ifngamma -/- , Tnf -/- , Gm-csf -/- , or Il-17 -/- mice according to schematic shown in B . Pooled data from two independent experiments with similar results ( n = 12-24 mice per group, as indicated). Log-rank tests ( Ifngamma -/- , Tnf -/- , Gm-csf -

Supportive validation

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Experimental details: Figure 3 Dynamics of Th1 cell population. A) Representative dot-plots of the population of CD4+Tbet+ cells of EAE animals. Dot-plots were obtained by previously gating in the CD3+ T cell population. Different quadrants were defined by application of the appropriate isotype control. A minimum of three animals per group was pooled and three replicates per score were analyzed. B and C) Histogrammes showing, respectively, the values corresponding to the total number and the percentage of CD4+Tbet+ cell population along EAE. Note that CD4+Tbet+ lymphocytes are found during the induction and peak phases and markedly decreased at score 2R of the recovery phase (ANOVA and Tukey's post-hoc test, *p

Supportive validation

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Experimental details: Figure 1 Homozygous RhoA deletion in Treg cells leads to early, fatal spontaneous inflammatory disorders. (A) Survival outcome of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. Results were analyzed with a log-rank (Mantel-Cox) test and expressed as Kaplan-Meier survival curves. (B) Image of lymphadenopathy in RhoA Flox/Flox Foxp3 YFP-Cre mice. Inguinal lymph nodes are shown. (C) Images of H&E staining of the indicated organs from RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice (original magnification X 400). (D) Left, representative flow cytogram of CD44 and CD62L staining in CD4 + and CD8 + cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. The numbers indicate percentages of CD44 + , CD44 + CD62L + , and CD62L + cells. Right, average percentages of CD44 + , CD44 + CD62L + , and CD62L + cells. (E) Left, representative flow cytogram of IL-17, IFN-gamma, and IL-4 staining in CD4 + Foxp3 - cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. The numbers indicate percentages of IL-17 + , IFN-gamma + , and IL-4 + cells. Right, average percentages of IL-17 + , IFN-gamma + , and IL-4 + cells. (F) Left, representative flow cytogram of RORgammaT, T-bet and GATA3 staining in CD4 + Foxp3 - cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. The numbers indicate percentages of RORgammaT + , T-bet + , and GATA3 + cells. Right, average percentages of RORgam

Supportive validation

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Experimental details: Figure 2 Homozygous RhoA deletion in Treg cells dampens Treg cell homeostasis and induces Treg cell plasticity. (A) Left, representative flow cytogram of Foxp3 staining in CD4 + cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. The numbers indicate percentages of CD4 + Foxp3 + Treg cells. Middle, average percentages of CD4 + Foxp3 + Treg cells. Right, average numbers of CD4 + Foxp3 + Treg cells. (B) Treg cell proliferation. Percentages of CD4 + Foxp3 + Treg cells incorporated with BrdU are shown. (C) Treg cell apoptosis. The expression levels (MFI: Mean fluorescence intensity) of active caspase 3 in CD4 + Foxp3 + Treg cells are shown. (D) The expression levels of Foxp3 in Treg cells. (E) Left, representative flow cytogram of IL-17, IFN-gamma, and IL-4 staining in CD4 + Foxp3 + Treg cells. The numbers indicate percentages of IL-17 + , IFN-gamma + , and IL-4 + Treg cells. Right, average percentages of IL-17 + , IFN-gamma + , and IL-4 + Treg cells. (F) Left, representative flow cytogram of RORgammaT, T-bet and GATA3 staining in CD4 + Foxp3 + Treg cells. The numbers indicate percentages of RORgammaT + , T-bet + , and GATA3 + Treg cells. Right, average percentages of RORgammaT + , T-bet + , and GATA3 + Treg cells. (G) Left, representative histogram of the expression levels of CTLA-4, GITR and PD-1 in CD4 + Foxp3 + Treg cells. The numbers above the graphs indicate MFI. Right, average MFI of CTLA-4, GITR and PD-1 in CD4 + Foxp3 + Treg cells. n =

Supportive validation

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Experimental details: Figure 5 Heterozygous RhoA deletion in Treg cells induces Treg cell plasticity and increases CD4 + effector T cells but does not result in autoimmunity. (A) Body weight of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/+ Foxp3 YFP-Cre mice. (B) Images of H&E staining of the indicated organs. (C) Representative flow cytogram of Foxp3 staining in CD4 + cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/+ Foxp3 YFP-Cre mice. The numbers indicate percentages of CD4 + Foxp3 + Treg cells. (D) Left, average percentages of CD4 + Foxp3 + Treg cells. Right, cell numbers of CD4 + Foxp3 + Treg cells. (E) Proliferation of Foxp3 + YFP + Treg cells from RhoA +/+ Foxp3 YFP-Cre/+ and RhoA Flox/+ Foxp3 YFP-Cre/+ female mice. Percentages of Foxp3 + YFP + Treg cells incorporated with BrdU are shown. (F) Apoptosis of Foxp3 + YFP + Treg cells from RhoA +/+ Foxp3 YFP-Cre/+ and RhoA Flox/+ Foxp3 YFP-Cre/+ female mice. The expression levels (MFI: mean fluorescence intensity) of active caspase 3 in Foxp3 + YFP + Treg cells are shown. (G) The expression levels of Foxp3 in Treg cells from RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/+ Foxp3 YFP-Cre mice. (H) Representative flow cytogram of IFN-gamma, IL-17 and IL-4 staining in CD4 + Foxp3 + Treg cells. The numbers indicate percentages of IFN-gamma + , IL-17 + and IL-4 + Treg cells. (I) Average percentages of IFN-gamma + , IL-17 + and IL-4 + Treg cells. (J) Representative flow cytogram of RORgammaT, T-bet and GATA3 staining in CD4 + Foxp3 + Treg cells. The numb

Supportive validation

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Experimental details: Figure 2 Effector CD4 T Cell Response to Adjuvanted Vaccines Groups of C57BL/6 mice were vaccinated IN, as in Figure 1 . At day 8 PV, cells from lungs and BAL were stained with I-A b /NP311 tetramers along with antibodies to cell surface molecules and transcription factors. (A) FACS plots show the percentages of I-A b /NP311 tetramer-binding cells among CD4 T cells. (B) Percentages of the indicated cell population among NP311-specific, tetramer-binding CD4 T cells. (C) FACS plots are gated on I-A b /NP311 tetramer-binding cells, and the numbers in each quadrant are the percentages of cells among the gated population; MFIs for transcription factors in NP311-specific CD4 T cells are plotted in the adjoining graphs. (D) FACS plots in (C) were used to quantify the percentages of T-bet LO EOMES HI cells (quadrant 4) among NP311-specific CD4 T cells. (E) Percentages of CD103 HI and CD69 HI cells among NP311-specific CD4 T cells. Data are representative of two independent experiments. Comparisons were made using one-way ANOVA test with Tukey-corrected multiple comparisons; *p < 0.05, **p < 0.01, and ***p < 0.001.

Supportive validation

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Experimental details: Fig. 9 In vivo administration of dNP2-siChi3l1 complex inhibits pulmonary metastasis with enhanced Th1 and CTL effector molecules. a Experimental scheme of dNP2-siRNA complex treatment in pulmonary melanoma metastasis model. b Representative lung image of dNP2-siEGFP and dNP2-siChi3l1-treated mice. Scale bar, 2 mm c Number of pleural colonies in each lung was counted. Data are mean +- SEM of three sets of independent experiments and each dot in graphs represent an individual mouse. d Relative total tumor area in the lung was measured by Image J software 1.48 v. e Histology of lung sections by H&E staining, and infiltrated tumor region was measured by Image J software 1.48 v. Scale bar, 200 m f , g IFNgamma producing CD4 and CD8 T cells, and Granzyme B expression level in IFNgamma + CD8 T cells in the lung was analyzed by intracellular staining. % of IFNgamma + , and MFI of Granzyme B was represented as scattered graph. h Intracellular T-bet expression level in CD4 + IFNgamma + and CD8 + IFNgamma + population. MFI was represented as scattered graph. i mRNA expression of genes related to cytotoxicity and Th1 effector functions was analyzed by quantitative RT-PCR. Each gene expression level was normalized to beta-actin. Data are mean +- SD of three sets of independent experiments and each dot in graphs represent an individual mouse. n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student's t -test)

Supportive validation

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Experimental details: Figure 2. Similarities between Infection-Induced Mouse FCRL5 + B Cells and atMBCs from Plasmodium -Infected Humans (A) RNA-seq was performed on class-switched (IgM - IgD - ), FCRL5 - or FCRL5 + B cells sorted from the blood of Ifngr1 - / - mice infected for 21 days with P. chabaudi . Columns represent individual mice (n = 5). Select genes involved in immune signaling are shown. Genes with putative activating function are labeled in green; those with putative inhibitory function are labeled in red. (B) Heatmaps depict all genes that are differentially regulated in both human atMBCs (left) and mouse FCRL5 + B cells, relative to cMBCs and FCRL5 - B cells, respectively. Human data are from . (C-E) Expression of (C) CD72 and CD38, (D) T-bet, and (E) CD11b, CD11c, CD86, and CD40 were measured by flow cytometry in blood B cells 21 d.p.i. **p < 0.01; ***p < 0.001 by paired t test (Wilcoxon rank-sum test). See also Figure S2 and Tables S1 and S2 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1. Characterization of CD4 + TIL, dLN, and Arm Transcriptomes by scRNA-Seq (A-D) TILs and dLN cells from wild-type (WT) mice at day 14 after MC38-GP injection analyzed by scRNA-seq and flow cytometry. (A) Heatmap shows row-standardized expression of selected genes across TIL and dLN clusters. Bar plot indicates the percentage of cells in each cluster relative to the total TIL or dLN cell number. (B) Flow cytometry contour plots of Foxp3 versus T-bet in CD44 hi GP66 + dLN cells (left) and in CD44 hi CD4 + splenocytes from tumor-free control mice (right). (C) Flow cytometry contour plots of Foxp3 versus T-bet in PD-1 + and GP66 + TILs (left) and in CD44 hi CD4 + splenocytes from tumor-free control mice (right). (B and C) Data representative from 18 tumor-bearing mice analyzed in four separate experiments. (D) t-SNE display of TILs and dLN cells, shaded gray by tissue origin (left) or color coded by main group (right, as defined in A). (E) t-SNE (TIL and dLN cell positioning as shown in B) display of normalized expression levels of selected genes. (F) Heatmap shows Pearson correlation between cluster fold change vectors (as defined in the text) across the two replicate experiments for TILs (left) and dLN cells (right). See also Figures S1 and S2 and Tables S1 and S6 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Characteristics of iKP-19-CAR-T cells and CD19-CAR-T cells. iKP-19-CAR-T cells or CD19-CAR-T cells were cultured for 10 days in X-VIVO media supplemented with 100 U/mL IL-2. ( A ) The expression of T cell differentiation markers in CAR-T cells was analyzed by flow cytometry using PE/Cy7-anti-human CCR7 antibody, PE-anti-human-CD45RO antibody and PE-anti-human GzmB antibody ( n = 4 different donors). ( B ) The frequency of naive (T N ; CCR7 + CD45RA + ), T CM (CCR7 + CD45RA - ), effector memory (T EM ; CCR7 - CD45RA - ) or effector (T E ; CCR7 - CD45RA + ) T cells were analyzed by flow cytometry using PE/Cy7-anti-human CCR7 antibody and FITC-anti-human CD45RA antibody ( n = 4 different donors). ( C ) The expression of transcription factor Eomes in CAR-T cells was analyzed by flow cytometry using FITC-anti-human Eomes antibody ( n = 4 different donors). ( D ) The expression of transcription factor T-bet in CAR-T cells was analyzed by flow cytometry using PE-anti-human T-bet antibody ( n = 4 different donors). ( E ) The expression of T cell exhaustion marker PD-1 in CAR-T cells was analyzed by flow cytometry using FITC-anti-human PD-1 antibody ( n = 4 different donors). All experiments were performed in triplicate manner using PBMCs from each donor and MFI or percentage was statistically analyzed. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars represent +- SD.