Journal: Oncoimmunology

Article Title: Dasatinib promotes Th1-type responses in granzyme B expressing T-cells

doi: 10.4161/onci.28925

Figure Lengend Snippet: Figure 1. The proportion of granzyme B positive (GrB+) T-cells is increased in CML patients at diagnosis and further expands during dasatinib therapy. Fresh or frozen PBMNCs were first stained for surface markers (α-CD45, α-CD3, α-CD4 and α-CD8), and after fixation and permeabilization intracellular GrB was stained, and cells were analyzed with flow cytometry. ( A ) The relative proportions of GrB+CD4+ T-cells and ( B ) GrB+CD8+ T-cells in samples obtained from healthy controls (n = 5) and CML patients at diagnosis (dg) (n = 18). ( C ) The proportion of GrB+CD4+ T-cells and ( D ) GrB+CD8+ T-cells 6 mo after start on dasatinib (DA, n = 7), imatinib (IM, n = 6), or nilotinib (NI, n = 6) therapy. The absolute number of GrB+CD4+ ( E ) and GrB+CD8+ T-cells ( F ) was measured in CML patients 6 mo after the start of DA (n = 8), IM (n = 6), or NI (n = 5) therapy. Panels A and B were analyzed by nonparametric Mann Whitney t test and panels C, D and E by 1way ANOVA.

Article Snippet: For the analysis of GrB+ T-cells, the cells were first stained for cell surface markers; α-CD45 APC-H7 (BD 641417), α-CD3 APC (BD 345767), α-CD4 PerCP (BD 345770), and α-CD8 PE-Cy7 (BD 335822), and then fixed and permeabilized with Fix/Perm (BD 554714), and intracellular GrB was stained (Alexa Fluor 700, BD 561016)).

Techniques: Staining, Flow Cytometry, MANN-WHITNEY

Journal: Journal of medicinal chemistry

Article Title: Aryl Trehalose Derivatives as Vaccine Adjuvants for Mycobacterium tuberculosis

doi: 10.1021/acs.jmedchem.9b01598

Figure Lengend Snippet: Induction of IL-17 producing CD4+ T cells from UM1024 adjuvanted M72 vaccine. BALB/c mice, 10 per group, were immunized two times i.m. with 0.1 μg antigen plus 2, 10 or 50 nmol of the indicated adjuvant. Spleens were harvested from 3 mice per group at 5 days post-secondary vaccination. Splenocytes were restimulated with 1 μg whole antigen and transport inhibitors followed by surface staining for CD3, CD4 and CD8 and intracellular cytokine staining for IL-17A. Data represent percentage of live, CD3+/CD4+ cells that are also positive for IL-17A upon antigen restimulation.

Article Snippet: Following incubation, cells were stained with the cell surface antibodies against CD3e PerCP-Cy5.5 (Tonbo Biosciences, 145–2C11), CD4a APC-Cy7 (Tonbo Biosciences, RM4–5) and CD8a PE-Cy7 (Tonbo Biosciences, 53–6.7) and viability stain (Ghost 510, Tonbo Biosciences).

Techniques: Adjuvant, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Identification and validation of T-cell receptors targeting RAS hot-spot mutations in human cancers for use in cell-based immunotherapy

doi: 10.1158/1078-0432.CCR-21-0849

Figure Lengend Snippet: A. Patient apheresis is collected. The blood is separated into adherent cells that are used to generate immature DC (imDC). The imDC are loaded with short predicted minimal epitopes (ME) or with long peptides (LP) or are transfected with mRNA of the full length (FL) gene of the specific RAS mutation or with tandem minigenes (TMG) containing all the common (more than 0.5%) RAS mutations. These Ag-loaded DC are used for activating and testing T cells in all stages. B. Non-adherent cells (PBL) are sorted into CD4 or CD8 T cells (CD3), central/effector memory (TCM/TEM), or effector (TEMRA) cells (excluding naïve T cells) by using CD45RO and CD62L. This subset is used for in vitro stimulation (IVS) with RAS Ag-loaded DC by coculture for three days with IL-21 and then expanded with media containing IL-21+IL-2 every two days. Cells are restimulated and sorted for high expression of 41BB/OX40 and rapidly expanded (REP) for another 14 days. C. Tumor metastasises are resected and cut into 24 fragments. Tumor-infiltrating lymphocytes (TIL) collected from each fragment are expanded in media containing IL-2 until there are enough cells for testing (usually about 4–6 weeks). D. T cells are tested for reactivity to RAS mutations by IFN-γ ELISpot and 41BB/OX40 flow cytometry assays. The positive cultures are sorted to enrich the RAS reactive cells and then single cells are sequenced. The TCRs are reconstructed (iTCR) using mouse constant TCR elements to enhance the iTCR α/β pairing. The iTCR are transduced into autologous/healthy donor PBLs and tested for specific RAS-mutated and WT reactivity. Specific RAS-mutated TCRs with good avidity could then be used to treat the patient (as neo-antigen treatment). E. After identifying the MHC restriction, the TCR could potentially be used to treat other patients having the same RAS mutation and MHC. All TCRs are saved in a library as prepared GMP vectors.

Article Snippet: CD3-APC-Cy7, CD4- PE/FITC/APC, 4–1BB- APC/PE, CD8- PE-Cy7/FITC, OX40- FITC/PE-Cy7, CD62L-PE, and CD45RO-APC/BV421 were purchased from BD (Becton Dickinson Biosciences).

Techniques: Transfection, Mutagenesis, In Vitro, Expressing, Enzyme-linked Immunospot, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Identification and validation of T-cell receptors targeting RAS hot-spot mutations in human cancers for use in cell-based immunotherapy

doi: 10.1158/1078-0432.CCR-21-0849

Figure Lengend Snippet: TIL fragments were screened for reactivity against autologous DC transfected with RAS TMG (WT/Mutated) or loaded with 24-mer long peptide (RASG12V/ RASWT). Coculture with DC loaded with DMSO was used as a negative control. Anti-CD28/CD3 beads were used as a positive control. A. Most fragments, after gating on the live/CD3+ cells and including fragment 1 (F1), primarily contained CD8+ cells. B. Fragment reactivity tested in flow cytometry assays measured upregulation of 41BB+ and OX40+ or C. by IFN-γ ELISpot. D. Table containing the patient 4391 MHC-I restriction elements. E. Table with the best-predicted RASG12V minimal epitopes (low % rank) to bind to the patient MHCs by the NetMHC 4.0. F. To determine which ME is recognized by TIL F1, cells were cocultured with autologous DC preloaded with RASG12V ME8, with LP (RASG12V/ RASWT), or with DMSO at an equivalent concentration. Reactivity was determined by upregulation of 41BB and OX40 surface markers in live/CD3+/CD8+ by flow cytometry. G. For testing the MHC-I restriction element, TIL F1 were cocultured with COS7 cells pre-transfected with patient’s class I HLA DNA plasmids and loaded with LP (RASG12V/RASWT) or ME8. Reactivity was determined by IFN-γ ELISpot. One TCR was identified from patient 4391 TIL fragment F1 after single-cell sequencing. This TCR was virally transduced to healthy donor (HD) PBLs. H. The TCR avidity was tested in the transduced cells by coculture with DC loaded with different concentrations of RASG12V/RASWT 9-mer ME8 or the equivalent WT sequence WT4. The cells were tested via IFN-γ ELISpot, with anti-CD28/CD3 beads used as a positive control. I. The TCR specificity and tumor recognition tested by coculturing the TCR transduced healthy donor (HD) cells with different tumor cell lines with or without (+/−) RASG12V and HLA-C*01:02. Tumor cell lines were loaded with RASG12V ME8, or not peptide-loaded (media). Unloaded tumor cell lines expressed their native peptidomes (including RASG12V). The reactivity was tested by IFN-γ ELISA and by flow cytometry assay for 41BB/OX40 surface marker upregulation in live/CD3+/mTCR+/CD8+ cells. Untransduced HD loaded with ME8 was used as a negative control.

Article Snippet: CD3-APC-Cy7, CD4- PE/FITC/APC, 4–1BB- APC/PE, CD8- PE-Cy7/FITC, OX40- FITC/PE-Cy7, CD62L-PE, and CD45RO-APC/BV421 were purchased from BD (Becton Dickinson Biosciences).

Techniques: Transfection, Negative Control, Positive Control, Flow Cytometry, Enzyme-linked Immunospot, Concentration Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Marker

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Identification and validation of T-cell receptors targeting RAS hot-spot mutations in human cancers for use in cell-based immunotherapy

doi: 10.1158/1078-0432.CCR-21-0849

Figure Lengend Snippet: PBL were sorted into CD4 or CD8 memory and effector T cells and in vitro stimulated (IVS) with DC loaded with RASG12V LP. After the stimulation period, T cells were restimulated with the same target cells. A. Flow cytometry assay dot plot showing the gating strategy in which CD4 cells were sorted for high expression of OX40 and 41BB surface markers following RASG12V LP IVS, with DMSO used as a negative control. B-C. The sorted cells were rapidly expanded (REP) following the IVS protocol (LP IVS), and were tested for reactivity against RAS by coculturing with DC transfected with WT/Mut TMG or loaded with RASG12V/RASWT LP or an equivalent amount of DMSO. Cells that were stimulated but didn’t sort (no sort) or unstimulated cells (no stimulation) were used as a control for the IVS. T cell only and with anti-CD28/CD3 beads used as negative and positive controls, respectively. After overnight coculturing, cells were analyzed for B. IFN-γ ELISpot and C. 41BB/OX40 surface marker upregulation in the live/CD3+/CD4+ gated population by flow cytometry analysis. D-E. CD4 cells after RASG12V LP IVS (including one REP after sorting) were tested for avidity to RASG12V. The cells were cocultured with DC loaded with RASG12V/ RASWT LP at various concentrations. After overnight coculturing, cells were analyzed via B. IFN-γ ELISpot and C. 41BB/OX40 surface marker upregulation in the live/CD3+/CD4+ gated population by flow cytometry analysis. F. The MHC-II restriction element recognized by 4360 CD4 PBL after RASG12V LP IVS was determined via IFN-γ ELISpot (left axis and bars) and 41BB/OX40 flow cytometry assay (right axis and circles). The cells were cocultured with COS7 transfected with DNA plasmids containing the different combinations of the patient’s MHC-II α and β chains and loaded with RASG12V LP.

Article Snippet: CD3-APC-Cy7, CD4- PE/FITC/APC, 4–1BB- APC/PE, CD8- PE-Cy7/FITC, OX40- FITC/PE-Cy7, CD62L-PE, and CD45RO-APC/BV421 were purchased from BD (Becton Dickinson Biosciences).

Techniques: In Vitro, Flow Cytometry, Expressing, Negative Control, Transfection, Control, Enzyme-linked Immunospot, Marker

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Identification and validation of T-cell receptors targeting RAS hot-spot mutations in human cancers for use in cell-based immunotherapy

doi: 10.1158/1078-0432.CCR-21-0849

Figure Lengend Snippet: A-K. Two TCRs (TCR1, TCR2) identified from patient 4360 CD4 PBL after LP IVS single-cell sequencing. A. Deep sequencing (Adaptive Biotechnologies) of DNA extracts from four tumor fragments (FrTu) revealed TCR1 existed in one of these fragments (5.7 repeats in 100,000 cells) while TCR2 did not exist in any. B. Both TCRs were mRNA transfected or C. virally transduced into a Jurkat-CD4-NFAT-Luciferase cell line and then cocultured with DC loaded with RASG12V/ RASWT LP or the equivalent amount of DMSO. Luciferase activity was measured and presented in Luminescence units. D-G. TCR1 and H-K. TCR2 were virally transduced into patient 4360 PBLs. The cells enriched to CD3+/CD4+ cells D, F, H, and J or enriched to CD3+/CD8+ E,G,I, and K. The transduced cells were cocultured with DC loaded with different concentration of RASG12V/ RASWT LP. TCR avidity in live/CD3+/mTCR+ cells was analyzed by D, E, H, I. flow cytometry for 41BB/OX40 surface marker upregulation and F, G, J, K. IFN-γ ELISpot.

Article Snippet: CD3-APC-Cy7, CD4- PE/FITC/APC, 4–1BB- APC/PE, CD8- PE-Cy7/FITC, OX40- FITC/PE-Cy7, CD62L-PE, and CD45RO-APC/BV421 were purchased from BD (Becton Dickinson Biosciences).

Techniques: Sequencing, Transfection, Luciferase, Activity Assay, Concentration Assay, Flow Cytometry, Marker, Enzyme-linked Immunospot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Identification and validation of T-cell receptors targeting RAS hot-spot mutations in human cancers for use in cell-based immunotherapy

doi: 10.1158/1078-0432.CCR-21-0849

Figure Lengend Snippet: Validated CD8 TCRs recognizing RAS hotspot mutations, available for allogeneic treatment. CD8 TCR table showing columns (from left to right): the hotspot mutation recognized by the TCR and the percentage of this mutation among RAS-mutated cancer patients. Number of TCRs discovered from the same patient recognizing the same target. The HLA-I restriction each TCR recognizes. Percentage of the allele frequency (taken from Allelefrequencies.net ) and calculation of the percentage of the individuals in the Caucasian or Asian populations (*). Percentage of patients eligible for treatment, calculated by multiplying the frequency of individuals that have the allele with the frequency of this RAS mutation (as in the first column) with the sum of percentages from all TCRs (the bottom of this column). ME sequence recognized by the TCR. Patient number. The method used to find the TCR (TIL screen/PBL IVS). Patient cancer diagnosis in which TCR was found. Reference if the TCR has been published before and, if so, where.

Article Snippet: CD3-APC-Cy7, CD4- PE/FITC/APC, 4–1BB- APC/PE, CD8- PE-Cy7/FITC, OX40- FITC/PE-Cy7, CD62L-PE, and CD45RO-APC/BV421 were purchased from BD (Becton Dickinson Biosciences).

Techniques: Mutagenesis, Sequencing

Journal: PLoS ONE

Article Title: Deletion of L-Selectin Increases Atherosclerosis Development in ApoE −/− Mice

doi: 10.1371/journal.pone.0021675

Figure Lengend Snippet: Immunohistochemical analysis of A ) macrophages (CD68 + ), B ) T-lymphocytes (CD3 + ), C ) T helper cells (CD4 + ), and D ) T-cytotoxic cells (CD8 + ) in aortic arch after 6 and 12 weeks of HCD (n = 5–13; p = n.s.).

Article Snippet: Blood cells were stained with fluorescently labeled anti-mouse CD4 (PE-conjugated, clone RM4-5), CD8 (PE-Cy7-conjugated, clone H35-17.2), CD19 (PE-Cy7-conjugated, clone 1D3), or CD44 (APC-conjugated, clone IM7) antibodies (Pharmingen) for 30 minutes at 4°C.

Techniques: Immunohistochemical staining

Journal: PLoS ONE

Article Title: Deletion of L-Selectin Increases Atherosclerosis Development in ApoE −/− Mice

doi: 10.1371/journal.pone.0021675

Figure Lengend Snippet: ApoE − / − L-sel − / − animals exhibit increased number of A ) total blood lymphocytes (n = 5–8; * p<0.05), B ) T-cytotoxic cells (CD8 + ; n = 6–7; ** p<0.01, # p<0.05), C ) B lymphocytes (CD19 + ; n = 5–7; * p<0.05, # p<0.05), D ) total T-helper cells (CD4 + ; n = 6–7; # p<0.0), E ) naive T-helper cells (CD4 + CD44 − ; n = 5–7; * p<0.05; ## p<0.0001) after 6 and 12 weeks of HCD, respectively. F ) The number of activated T-cells (CD4 + CD44 + ) remained unchanged after 6 weeks of HCD (n = 5–7; p = n.s.) and decreased after 12 weeks of HCD (n = 5–7; * p<0.05) as compared to control.

Article Snippet: Blood cells were stained with fluorescently labeled anti-mouse CD4 (PE-conjugated, clone RM4-5), CD8 (PE-Cy7-conjugated, clone H35-17.2), CD19 (PE-Cy7-conjugated, clone 1D3), or CD44 (APC-conjugated, clone IM7) antibodies (Pharmingen) for 30 minutes at 4°C.

Techniques: