mixed anti human cd4  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Cis-Regulatory Atlas in Primary Human CD4+ T Cells

doi: 10.1101/2022.12.09.519788

Figure Lengend Snippet: MACS2 Log2FC (for enhancers) and FAST-NR Log2FC (for NREs) are displayed in the genome browser above. Fragment Tested represents the DNA sequence cloned for luciferase testing. (*) Non-overlapping 95% Confidence Intervals. Error represented as Mean ± SD. A) Twenty putative NREs are tested using CMV-Enhancer-LUC in DualGLO Luciferase assays (Promega). Two independent human CD4+ Donors were used for each CRE tested, with two technical replicates each. Relative Activity compares NRE activity to empty-vector CMV-Enhancer-LUC control activity. B) Twenty putative enhancers are tested using pGL4.26 (minP promoter) in DualGLO Luciferase assays as described above. C) Binomial exact test trials to estimate the true fraction of tested CREs that are functional in luciferase assays. P-value indicates the likelihood that the observed proportion of luciferase active fragments is greater than the theoretical proportion on the x-axis.

Article Snippet: The STARR-Seq inset library was created according to the ATAC-STARR-Seq approach. ( ) Briefly, OMNI ATAC-Seq was performed as previously described with 320,000 human CD4+ T cells (in total) in batches of 100,000 cells per reaction (NEB #M0544S). ( ) The ATAC-Seq reaction products were cleaned up using the Qiagen Reaction Cleanup Kit (Qiagen #28206) and eluted in 12 μL per 100,000 cells transposed.

Techniques: Sequencing, Clone Assay, Luciferase, Activity Assay, Plasmid Preparation, Functional Assay

Journal: bioRxiv

Article Title: Cis-Regulatory Atlas in Primary Human CD4+ T Cells

doi: 10.1101/2022.12.09.519788

Figure Lengend Snippet: A) PRO-Seq tags in hCD4+ T cells (GSE66031) at STARR-Seq Enhancers, NREs, and ‘All’ open peaks from input plasmid library. B) ChIP-Seq experiments of RNA Polymerase II Phospho-S5 and total RNA Polymerase II conducted in human CD4+ T cells from Barski et al . 2007 C) Chromatin looping targets of CRE to promoter assigned from Javierre 2016, with TPM from hCD4+ PolyA RNA-Seq (ENCSR545MEZ). (** P-value < 0.01 by non-parametric Kruskal-Wallis two-sided test with Holm adjustment for multiple comparisons). D) Number of CRE:Promoter PC-HiC contacts to each target gene, with TPM from hCD4+ PolyA RNA-Seq (ENCSR545MEZ). E) Multivariate linear model explaining TPM (ENCSR545MEZ) by number of CRE:Promoter interactions to each promoter [TPM ~ ß ATAC + ß ENH + ß NREs ]. ß slope coefficients ± Standard Error displayed. (** P-value < 0.001)

Article Snippet: The STARR-Seq inset library was created according to the ATAC-STARR-Seq approach. ( ) Briefly, OMNI ATAC-Seq was performed as previously described with 320,000 human CD4+ T cells (in total) in batches of 100,000 cells per reaction (NEB #M0544S). ( ) The ATAC-Seq reaction products were cleaned up using the Qiagen Reaction Cleanup Kit (Qiagen #28206) and eluted in 12 μL per 100,000 cells transposed.

Techniques: Plasmid Preparation, ChIP-sequencing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Cis-Regulatory Atlas in Primary Human CD4+ T Cells

doi: 10.1101/2022.12.09.519788

Figure Lengend Snippet: RELI enrichment logP-value of CRE enrichment across available transcription factor ChIP-Seq experiments in GEO. Heatmaps of RNA expression [transcripts per million (TPM)] in resting human CD4+ T cells are displayed furthest left. Transcription factor family is displayed adjacent to RNA expression. A) The top 2500 ChIP-Seq experiments with strongest enrichment in STARR-Seq enhancers are displayed, whereas B) displays the top experiments with NRE enrichment. C) Motif enrichment performed in HOMER de novo motifs in Enhancers (predicted motif, p-value, and transcription factor name) and D) in NREs.

Article Snippet: The STARR-Seq inset library was created according to the ATAC-STARR-Seq approach. ( ) Briefly, OMNI ATAC-Seq was performed as previously described with 320,000 human CD4+ T cells (in total) in batches of 100,000 cells per reaction (NEB #M0544S). ( ) The ATAC-Seq reaction products were cleaned up using the Qiagen Reaction Cleanup Kit (Qiagen #28206) and eluted in 12 μL per 100,000 cells transposed.

Techniques: ChIP-sequencing, RNA Expression

Journal: PLoS ONE

Article Title: Effects of Exogenous Recombinant APC in Mouse Models of Ischemia Reperfusion Injury and of Atherosclerosis

doi: 10.1371/journal.pone.0101446

Figure Lengend Snippet: Images of plaques stained for collagen ( A ), macrophages ( B ), leukocytes ( C ), and T cells ( D ). E) After sirius red staining, the percentage of collagen was determined by dividing the area of collagen by the total plaque area. F) After Mac-3 staining, the percentage of macrophages as a percentage of the total number of cells per plaque was calculated. G) After CD45 staining, the percentage of leukocytes as a percentage of the total number of cells per plaque was calculated. H) After CD3 staining, the percentage of T cells of the total number of cells per plaque was calculated. All graphs show mean +/− SEM, statistical significance was tested using one-way analysis of variance with Dunnett post hoc test, *P<0.05, **P<0.01.

Article Snippet: Blood and spleen were collected, processed and fluorescently stained with either antibody mix 1 (CD3-FITC, CD4-PerCp, CD8-eFLUO450, CD25-APC, Foxp3-PE and B220-PE-Cy7) or antibody mix 2 (CD11b-PE-Cy7, Ly6C-APC, Ly6G-PE, CD3-FITC, B220-APC-eFLUO780 and NK1.1-PerCp-Cy5.5) or isotype control IgG (all BD Biosciences or eBioscience).

Techniques: Staining

Journal: Molecular Therapy Oncolytics

Article Title: Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model

doi: 10.1016/j.omto.2021.12.019

Figure Lengend Snippet: Evaluation of anti-CAIX BBζ CAR-T cells in vitro (A) Designs of anti-CAIX BBζ, 28ζ, and 28BBζ CAR-T cells. The same anti-CAIX G36 scFv was used as CAR moiety followed by CD8 hinge, costimulatory domain (s), and activation cassette CD3ζ. ZsGreen was introduced after IRES. (B) Cytotoxicity of anti-CAIX BBζ CAR-T cells with different CD4:CD8 composition ratios. From left to right, the cytotoxicity of CD4, CD4:CD8 = 2:1, CD4:CD8 = 1:1, CD4:CD8 = 1:2, and CD8 anti-CAIX BBζ CAR-T against CAIX-expressing skrc-59 cells is shown. P values are defined by unpaired two-tailed t -tests ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). (C) Heatmap of log2 fold change cytokine and chemokine profiles in supernatant. After 48 h co-culture of skrc-59 tumor cells and CAR-T cells with different CD4:CD8 composition ratios, the media were assessed for cytokine and chemokine release. The fold change was defined by the value of experimental group divided by the one of UNT negative control. All values of fold change are labeled in the heatmap.

Article Snippet: In contemporary trials, patients are typically administrated with CD4/CD8 mixed CAR-T cell products, including Yescarta and Kymriah with a fixed CD4/8 ratio of 1 and Tecartus (brexucabtagene autoleucel) with a median CD4/8 ratio of 0.7. , , In this study, we observed the more rapid expansion of CD8 BBζ CAR-T cells in the CD4/8 mixture in vivo , resulting in the CD4/8 ratio changing from 2:1 to about 1:1.

Techniques: In Vitro, Activation Assay, Expressing, Two Tailed Test, Co-Culture Assay, Negative Control, Labeling

Journal: Molecular Therapy Oncolytics

Article Title: Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model

doi: 10.1016/j.omto.2021.12.019

Figure Lengend Snippet: Anti-CAIX BBζ CAR4/8 T cells had superior activity in ccRCC orthotopic NSG-SGM3 mouse model (A) Timeline of the experiment. A week after tumor engraftment, BLI was performed to confirm tumor growth followed by CAR-T cell injection (day 0). BLI and bleeding were performed weekly to monitor tumor growth and CAR-T persistence. MRI was performed biweekly to monitor tumor growth and metastasis. (B) Tumor growth curve of the mice treated with 3 million of anti-CAIX BBζ (pink curve), 28ζ (green curve), and 28BBζ (orange curve) CAR-T cells and UNT (black curve) cells. n.s., not significant. P values are defined by ANOVA ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). (C) Tumor weight of anti-CAIX BBζ CAR-T cells (pink circle) compared with 28ζ (green circle) and 28BBζ (orange circle) CAR-T cells and UNT (black curve). P values are defined by unpaired two-tailed t -tests ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). (D) Tumor growth curve of the mice treated with 3 million of anti-CAIX BBζ CAR8 (pink curve), BBζ CAR4/8 (blue curve), UNT CD8 (black curve), and UNT CD4/8 (gray curve) cells. P values are defined by ANOVA ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). (E) Tumor weight of CAR8 (pink curve) and CAR4/8 (blue curve) of BBζ CAR-T cells compared with the CD8 (black curve) and CD4/8 (gray curve) of UNT cells. P values are defined by unpaired two-tailed t -tests ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). Note that the endpoint of BBζ CAR4/8 group is day 72 instead of day 28. (F) BLI images of mice treated with 3 million of BBζ, 28ζ, 28BBζ, BBζ CAR4/8, UNT CD4/8, and UNT CD8 cells are shown here. (G) Day 0 (D0), D14, and D28 of MRI on BBζ-, 28ζ-, 28BBζ-, BBζ-CAR4/8-, UNT-CD4/8-, and UNT-CD8-treated mice. Red arrow indicates the tumor site. All data with error bars are presented as mean ± SD.

Article Snippet: In contemporary trials, patients are typically administrated with CD4/CD8 mixed CAR-T cell products, including Yescarta and Kymriah with a fixed CD4/8 ratio of 1 and Tecartus (brexucabtagene autoleucel) with a median CD4/8 ratio of 0.7. , , In this study, we observed the more rapid expansion of CD8 BBζ CAR-T cells in the CD4/8 mixture in vivo , resulting in the CD4/8 ratio changing from 2:1 to about 1:1.

Techniques: Activity Assay, Injection, Two Tailed Test

Journal: Molecular Therapy Oncolytics

Article Title: Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model

doi: 10.1016/j.omto.2021.12.019

Figure Lengend Snippet: Flow cytometry analysis of peripheral circulating and tumor-infiltrating CAR-T cells (A) T cell count in peripheral blood. Peripheral blood was analyzed weekly via flow cytometry. T cell count per 100 μL was quantified in BBζ CAR8 (pink), 28ζ CAR8 (green), 28BB CAR8 (orange), and BBζ CAR4/8 (blue) CAR-T cells and CD8 (black) and CD4/8 (gray) UNT cells. (B) Exhaustion of peripheral circulating T cells. The percentage of PD-1 high-expressing T cells was quantified in BBζ CAR8 (pink), 28ζ CAR8 (green), 28BB CAR8 (orange), and CD8 (black) UNT cells on day 7. P values are defined by unpaired two-tailed t -tests ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001). (C) Tumor-infiltrating T cell percentage. At the endpoint of the animal study, tumor-infiltrating human CD45 + lymphocytes were isolated. T cell infiltration of BBζ (pink), 28ζ (green), 28BBζ (orange), and BBζ CD4/8 (blue) CAR-T cells and CD8 (black) and CD4/8 (gray) UNT cells in tumors was quantified. The percentage was defined by human CD45 population divided by total viable cells. (D) Percent CD4 and CD8 TILs. (E) CAR + percentage. (F and G) Exhaustion marker expression (PD-1 high; F) and the memory T cell subset (CD45RO + CD45RA − ; G) of CAR4/8 BBζ CAR-T cells on day 72 were analyzed. ZsGreen signal was used to define CAR + population. P values are defined by unpaired two-tailed t -tests ( ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001).

Article Snippet: In contemporary trials, patients are typically administrated with CD4/CD8 mixed CAR-T cell products, including Yescarta and Kymriah with a fixed CD4/8 ratio of 1 and Tecartus (brexucabtagene autoleucel) with a median CD4/8 ratio of 0.7. , , In this study, we observed the more rapid expansion of CD8 BBζ CAR-T cells in the CD4/8 mixture in vivo , resulting in the CD4/8 ratio changing from 2:1 to about 1:1.

Techniques: Flow Cytometry, Cell Counting, Expressing, Two Tailed Test, Isolation, Marker

Journal: Molecular Therapy Oncolytics

Article Title: Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model

doi: 10.1016/j.omto.2021.12.019

Figure Lengend Snippet: scRNA-seq analysis of tumor-infiltrating T cells (A) t-SNE plot of tumor-infiltrating BBζ CAR8 (pink), 28ζ CAR8 (green), and 28BBζ CAR8 (orange) cells and CD8 UNT (black) and CD4/8 UNT (gray) cells. CD4 and CD8 gene distribution is shown in the lower t-SNE plots. (B) CD8 regulatory T cell (Treg cell) subset analysis of the five treatments by determining FOXP3 and IL-2RA (CD25) gene expression. FOXP3 − IL-2RA − (gray), FOXP3 − IL-2RA + (brown), FOXP3 + IL-2RA − (orange), and FOXP3 + IL-2RA + (yellow) distribution is shown in the t-SNE plot. (C and D) Quantification of the percentage (C) and cell count (D) of each population.

Article Snippet: In contemporary trials, patients are typically administrated with CD4/CD8 mixed CAR-T cell products, including Yescarta and Kymriah with a fixed CD4/8 ratio of 1 and Tecartus (brexucabtagene autoleucel) with a median CD4/8 ratio of 0.7. , , In this study, we observed the more rapid expansion of CD8 BBζ CAR-T cells in the CD4/8 mixture in vivo , resulting in the CD4/8 ratio changing from 2:1 to about 1:1.

Techniques: Expressing, Cell Counting

Journal: Molecular Therapy Oncolytics

Article Title: Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model

doi: 10.1016/j.omto.2021.12.019

Figure Lengend Snippet: Transcriptional signatures of tumor-infiltrating CD8 UNT and CD4/8 UNT cells (A) Heatmap of top 50 differential expression genes in the comparison between CD8 UNT (black) and CD4/8 (gray) UNT cells. (B) Gene expression of HLA-DRB1, KLF6, IL-7R, GZMB, NR4A2, and JUN in CD8 (black) and CD4/8 (gray) UNT cells. (C) Heatmap of top 72 differential expression genes in the comparison between CD8 T cells in CD8 UNT (black) and the ones in CD4/8 (gray) UNT cells.

Article Snippet: In contemporary trials, patients are typically administrated with CD4/CD8 mixed CAR-T cell products, including Yescarta and Kymriah with a fixed CD4/8 ratio of 1 and Tecartus (brexucabtagene autoleucel) with a median CD4/8 ratio of 0.7. , , In this study, we observed the more rapid expansion of CD8 BBζ CAR-T cells in the CD4/8 mixture in vivo , resulting in the CD4/8 ratio changing from 2:1 to about 1:1.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Bone marrow transplantation generates T cell–dependent control of myeloma in mice

doi: 10.1172/JCI98888

Figure Lengend Snippet: MM-bearing recipients were lethally irradiated and transplanted with 10 × 106 BM cells with or without 5 × 106 T cells from naive or myeloma-experienced donors. Mice were monitored for survival and tumor burden using M-band (G/A) levels. M-band levels were modeled to calculate a predictive rate of tumor growth (solid line), with shaded CIs and the M-band relapse threshold shown as a dotted line. (A) Tumor burden and survival of Vk12653-bearing recipients (n = 30; combined from 5 experiments) and (B) survival of Vk12598-bearing recipients (n = 10; combined from 2 experiments) that received TCD-BMT or BM and T cells from naive donors (BMT). (C) Tumor burden and survival of Vk12653-bearing recipients (n = 16–19 combined from 3 experiments) and (D) survival of Vk12598-bearing recipients (n = 12 combined from 2 experiments) transplanted with TCD-BMT, BMT, or TCD-BM with myeloma-experienced T cells (BMT+). (E) Tumor burden and survival of Vk12653-bearing recipients treated with saline or CD8- or CD4-depleting Abs (αCD8, αCD4) from day 0 to 8 weeks after BMT (n = 11 combined from 2 experiments). (F) Survival of Vk12653-bearing recipients of BMT grafts from NK cell–intact (Mcl1fl/fl or WT) or NK cell–deficient (NKp46CreMcl1fl/fl) donors (n = 18 combined from 2 experiments). To determine statistical significance, the tumor burden was plotted using longitudinal mixed-effects linear models, and survival was analyzed using a log-rank test. *P < 0.05 and ***P < 0.001.

Article Snippet: BM was depleted of T cells (TCD) using an Ab master mix against CD4, CD8, and CD90.2 and rabbit complement (Cedarlane Laboratories Ltd.) as described previously ( 72 ).

Techniques: Irradiation, Saline

Journal: The Journal of Clinical Investigation

Article Title: Bone marrow transplantation generates T cell–dependent control of myeloma in mice

doi: 10.1172/JCI98888

Figure Lengend Snippet: (A) Representative FACS plots and frequency of TCM (CD44+CD62L+) and TEM/EFF (CD44+CD62L–) cells in naive and myeloma-experienced donor grafts (n = 3 per group). (B–F) MM-bearing recipients were lethally irradiated and transplanted with 10 × 106 TCD-BM cells alone (TCD-BMT) or 3 × 106 CD44+ or CD44– T cells from CD45.1/CD45.2 myeloma-experienced donors. (B) Tumor burden, quantified and modeled using M-band levels as described, and survival of Vk12653-bearing recipients (n = 14–16 combined from 2 experiments). (C and D) Recipients were sacrificed 2 weeks after BMT+, and BM T cells were analyzed by flow cytometry (n = 5 per group from 1 experiment). (C) Absolute numbers of donor CD8+ and CD4 + T cells and TCM and TEM/EFF CD8+ T cells in BM. (D) Representative FACS plots and absolute numbers of DNAM-1+PD-1+ and exhausted (DNAM-1–PD-1+TIM-3+) donor CD8+ T cells. (E and F) Recipients of BMT+-CD44+ grafts were sacrificed more than 100 days after BMT+, and BM T cells were analyzed via flow cytometry (n = 6 from 1 experiment). (E) Absolute numbers of donor CD8+ and CD4+ T cells. (F) Representative FACS plot and absolute numbers of TCM and TEM/EFF donor CD8+ T cells. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by log-rank test for survival data and Mann-Whitney U test for 2-sample and ANOVA for multiple-sample comparisons.

Article Snippet: BM was depleted of T cells (TCD) using an Ab master mix against CD4, CD8, and CD90.2 and rabbit complement (Cedarlane Laboratories Ltd.) as described previously ( 72 ).

Techniques: Irradiation, Flow Cytometry, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Bone marrow transplantation generates T cell–dependent control of myeloma in mice

doi: 10.1172/JCI98888

Figure Lengend Snippet: MM-bearing recipients were transplanted with TCD-BM from naive mice and either naive T cells (naive) or T cells from MM-bearing recipients with long-term control of Vk12653 myeloma (>120 days after BMT: MM-exp). (A) Tumor burden, quantified and modeled using M-band levels as described, and survival of Vk12653-bearing recipients transplanted with either naive or MM-exp T cells (n = 14–18 combined from 2 experiments). (B) Representative FACS plots and frequency of TEM/EFF, TCM, and naive CD8+ T cells and (C) representative histograms and frequency of CD122+ cells within TCM CD8+ T cells in the BM of recipients of naive or MM-exp T cells, 120 days after BMT (naive, n = 4; MM-exp, n = 12 from 1 experiment). (D) Survival of Vk12653-bearing recipient mice and Vk12598-bearing recipient mice transplanted with naive TCD-BM and either naive T cells or MM-expT cells from mice with long-term control of Vk12653 myeloma (n = 10 combined from 2 experiments). (E) M-band 6 weeks after BMT of secondary recipients of naive TCD-BM and either naive T cells or 2 × 106 MM-exp CD8+ or 2 × 106 MM-exp CD4+ T cells transferred with naive CD4+ or CD8+ T cells, respectively (n = 10–11 combined from 2 experiments). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by Mann-Whitney U test for numerical values and log-rank test for survival data.

Article Snippet: BM was depleted of T cells (TCD) using an Ab master mix against CD4, CD8, and CD90.2 and rabbit complement (Cedarlane Laboratories Ltd.) as described previously ( 72 ).

Techniques: Control, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Bone marrow transplantation generates T cell–dependent control of myeloma in mice

doi: 10.1172/JCI98888

Figure Lengend Snippet: Adoptively transferred CD8+ T cells (CD45.2) were purified from the spleens of Vk12653-bearing mice with controlled disease (MM-controlled) and MM-free transplant control mice (MM-free) for TCRβ deep sequencing (n = 5 per group). (A) Percentage of the TCRβ repertoire based on the frequency of increasing numbers of clonotypes. (B and C) Distribution of rare, small, medium, large and hyperexpanded clonotypes in the TCRβ repertoire. (D) Entropy and (E) evenness of the TCRβ repertoire in rare clonotypes. (F) Total unique CDR3 sequences identified in 50,000, 100,000, 250,000, 500,000, and 750,000 sampled sequences. Residue-identical TCRβ clonotype overlap in (G) rare, (H) small, and (I) medium T cell subsets (n = 5 pairs of mice in each cohort). Data represent the mean ± SEM. *P < 0.05, ** P < 0.01, and ***P < 0.001, by unpaired t test.

Article Snippet: BM was depleted of T cells (TCD) using an Ab master mix against CD4, CD8, and CD90.2 and rabbit complement (Cedarlane Laboratories Ltd.) as described previously ( 72 ).

Techniques: Purification, Control, Sequencing, Residue

Journal: The Journal of Clinical Investigation

Article Title: Bone marrow transplantation generates T cell–dependent control of myeloma in mice

doi: 10.1172/JCI98888

Figure Lengend Snippet: (A) Tumor burden, quantified and modeled using M-band levels as described, and survival in MM-bearing recipients transplanted with BM and T cells from WT or IL-17A–deficient (IL-17A–/–) donors (n = 20 combined from 4 experiments). (B) M-band of MM-bearing BMT+ with myeloma-experienced donors that were treated twice a week with 150 μg i.p of an IL-17A–blocking Ab or isotype control (cIg) from day 0 to week 6 after BMT+ (n = 12–13 combined from 2 experiments). (C–E) MM-free and MM-bearing recipient mice were transplanted with BM and T cells from IL-17ACreRosa26eYFP donors. eYFP+ cells were analyzed in BM (femur) and spleens 6 weeks after BMT. Representative dot plot (concatenated BM) and (C) total eYFP+ frequency and numbers, (D) frequency of TCRγδ+ and TCRβ+ cells within the eYFP+ cell population, and (E) total numbers of Th17 (eYFP+TCRβ+CD4+) and Tc17 (eYFP+TCRβ+CD8+) cells (n = 9 combined from 2 experiments). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by log-rank test for survival and Mann-Whitney U test for numerical values.

Article Snippet: BM was depleted of T cells (TCD) using an Ab master mix against CD4, CD8, and CD90.2 and rabbit complement (Cedarlane Laboratories Ltd.) as described previously ( 72 ).

Techniques: Blocking Assay, Control, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Bone marrow transplantation generates T cell–dependent control of myeloma in mice

doi: 10.1172/JCI98888

Figure Lengend Snippet: (A–C) MM-bearing or MM-free B6.WT mice were transplanted as previously described, and IFN-γ production was determined ex vivo 8 weeks later. (A) Representative histograms, (B) geometric MFI, and (C) absolute numbers of IFN-γ–producing CD8+ T cells in mice with active myeloma progression (MM-relapsed) or controlled disease (MM-controlled) and in transplant control mice (MM-free) (MFI, n = 5–10 from 2 experiments; absolute number, n = 7–12 combined from 3 experiments). (D) Tumor burden and survival of MM-bearing recipient mice transplanted with BM and T cells from B6.WT, B6.IFN-γ–/–, or B6.IFN-γR–/– donors (n = 20 combined from 4 experiments). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by log-rank test for survival data and Mann-Whitney U test for 2-sample and ANOVA for multiple-sample comparisons.

Article Snippet: BM was depleted of T cells (TCD) using an Ab master mix against CD4, CD8, and CD90.2 and rabbit complement (Cedarlane Laboratories Ltd.) as described previously ( 72 ).

Techniques: Ex Vivo, Control, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Bone marrow transplantation generates T cell–dependent control of myeloma in mice

doi: 10.1172/JCI98888

Figure Lengend Snippet: MM-bearing recipient mice were transplanted with BM and T cells from B6.WT donors. Agonistic CD137 (clone 3H3) or a control mAb (IgG2a) was administered from week 2 to week 6 after BMT. (A) Illustration of experimental design. (B) Tumor burden, quantified using M-band levels as described, and (C) overall survival (n = 18 combined from 2 experiments). (D–G) BM and spleens were harvested after treatment ceased, and cells were analyzed using flow cytometry (n = 5–10 from 1 to 2 experiments). (D) Percentage of CD138+CD19– myeloma cells in BM. (E) Absolute numbers of CD3+, CD8+, and CD4+ T cells and frequency of FoxP3+ within CD4+ T cells in BM and spleens. (F) Representative histograms show KLRG1 expression in CD8+ T cells and absolute numbers of KLRG1+ cells within CD8+ and CD4+ T cells in BM. (G) Representative histograms show GrB, IFN-γ, and CD107a expression in CD8+ T cells and graphs show absolute numbers of GrB+, IFN-γ+, and CD107a+CD8+ T cells and GrB+ and IFN-γ+CD4+ T cells in BM. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by log-rank test for survival data and Mann-Whitney U test for numerical values.

Article Snippet: BM was depleted of T cells (TCD) using an Ab master mix against CD4, CD8, and CD90.2 and rabbit complement (Cedarlane Laboratories Ltd.) as described previously ( 72 ).

Techniques: Control, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Bone marrow transplantation generates T cell–dependent control of myeloma in mice

doi: 10.1172/JCI98888

Figure Lengend Snippet: MM-bearing recipient mice were transplanted with naive TCD-BM and T cells from myeloma-experienced donors. Agonistic CD137 (clone 3H3) or a control mAb (IgG2a) was administered from week 1 to week 5 after BMT+. (A) Tumor burden, quantified using M-band levels as described (n = 10 combined from 2 experiments). (B–F) BM was harvested after treatment ceased, and cells were analyzed using flow cytometry (n = 8–10, from 1 experiment). (B) Absolute numbers of CD8+ T and CD4+ T cells in BM. (C) Absolute numbers of KLRG1+, Ki67+, GrB+, IFN-γ+, TNF+, and CD107a+CD8+ T cells in BM. (D) tSNE analysis and heatmaps of median CD8+ T cell exhaustion marker coexpression. (E) CD8+ T cell exhaustion marker quantification. (F) Experimental design and M-band levels of MM-bearing recipients transplanted with 1 × 106 myeloma-experienced T cells and 10 × 106 naive TCD-BM cells and treated with CD137 mAb alone (from day 7), anti–PD-1 alone (from day 17), CD137 mAb plus anti–PD-1, or isotype control (cIg). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by Mann-Whitney U test for 2-sample and ANOVA for multiple-sample comparisons.

Article Snippet: BM was depleted of T cells (TCD) using an Ab master mix against CD4, CD8, and CD90.2 and rabbit complement (Cedarlane Laboratories Ltd.) as described previously ( 72 ).

Techniques: Control, Flow Cytometry, Marker, MANN-WHITNEY