Journal: bioRxiv

Article Title: Immune Checkpoint Molecules as Biomarkers of Staphylococcus aureus Bone Infection and Clinical Outcome

doi: 10.1101/2024.12.30.630837

Figure Lengend Snippet: (A) Immunofluorescent histochemistry analyses of tibia sections from uninfected and MRSA-infected humanized BLT mice 14 days post-op were performed with labeled antibodies against CD3, LAG-3, TIM-3, and PD-1 with DAPI counter stain, and representative images are shown at 4x. Note the increased numbers of T cells near the SAC (dashed yellow line) in the infected tibiae. (B) A multichromatic spectral flow cytometry analyses were performed on tibial bone marrow cells from uninfected and MRSA-infected BLT mice. Live human CD45+/CD3+/T cells and their subpopulations (CD4+, CD8+, Tregs) were analysed for immune checkpoint expression (LAG3, TIM-3, and PD-1) and proliferation (Ki67), and representative histograms are shown. Note the frequency of human CD3 + /CD4 + T cells expressing TIM-3, LAG3 & PD-1) in the cells from MRSA-infected bone marrow (n=4-8 mice, *p<0.05, t-test).

Article Snippet: Primary antibodies: The following antibodies were utilized for immunostaining: goat anti-CD3ε (clone M-20, sc-1127, RRID:AB_631128, Santa Cruz Biotechnology), mouse anti-PD-1 (10377-MM23, RRID:AB_2936309, Sino Biologicals), Rabbit anti-LAG3 (clone BLR027F, NBP2-76402, RRID:AB_3403543, Novus Biologicals), Mouse anti-TIM3/HAVCR2 (clone TIM3/4031, V8754-20UG, NSJ Bioreagents), Rabbit anti- S. aureus (PA1-7246, RRID:AB_561546, Thermo Fisher Scientific), and Mouse anti-CD66b (G10F5, NBP2-80664, RRID:AB_3096017, Novus Biologicals).

Techniques: Infection, Labeling, Staining, Flow Cytometry, Expressing

Journal: Immunology

Article Title: Functional exhaustion of CD4 + T cells induced by co‐stimulatory signals from myeloid leukaemia cells

doi: 10.1111/imm.12665

Figure Lengend Snippet: Expression of activation‐ and/or exhaustion‐associated molecules on CD4+ T cells incubated with myeloid leukaemia (ML) cells under aCD3 stimulation. (a) Purified CD4+ T cells were co‐cultured with HL‐60 or THP‐1 cells (at a 2 : 1 leukaemia cell : T‐cell ratio, 25 ng/ml aCD3) and the expression of CD25, CD127, inducible T‐cell co‐stimulator (ICOS), CD69, T‐cell immunoglobulin and mucin domain‐containing protein 3 (TIM‐3), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD‐1) and cytotoxic T‐lymphocyte antigen 4 (CTLA‐4) markers were determined on T cells at 0, 6, 12 and 24 hr of incubation (transient‐stimuli co‐culture). The co‐cultures were established (b) at a 2 : 1 leukaemia cell : T‐cell ratio in the presence of different concentrations of aCD3 or (c) at different leukaemia cell : T‐cell ratios in the presence of a constant aCD3 concentration (25 ng/ml), the antibody was refreshed together with the media for every 24 hr, and after 96 hr T cells were analysed by flow cytometry (continuous‐stimuli co‐culture), (n ≥ 3). (d) Expression of the inhibitory receptors on T cells from the 96 hr co‐cultures with different myeloid leukaemia ratios are shown. Representative overlay histograms out of three independent experiments are given. Iso. control, staining with isotype‐matched control antibody.

Article Snippet: The monoclonal antibodies anti‐human‐CD4 (SK3), ‐CD3 (HIT3a), ‐CD69 (FN50), ‐CD25 (M‐A251), ‐CD14 (M5E2), ‐CD13 (L138), ‐CD274 (PD‐L1; MIH1) (Becton Dickinson, San Jose, CA); ‐LAG3 (FAB2319F) (R&D, Minneapolis, MN); ‐CD154 (24–31), ‐CD127 (hIL‐7r‐M21), ‐CD80 (2D10), ‐CD86 (IT2.2), ‐CD152 (CTLA‐4; L3D10), ‐CD275 (ICOS‐LG; 9F.8A4), ‐CD278 (ICOS; C398.4A), ‐CD279 (PD‐1; EH12.2H7), ‐FoxP3 (236A/E7), ‐TIM‐3 (F38‐2E2) (Biolegend, San Diego, CA) were used in immunophenotyping analyses.

Techniques: Expressing, Activation Assay, Incubation, Purification, Cell Culture, Co-Culture Assay, Concentration Assay, Flow Cytometry, Staining

Journal: Immunology

Article Title: Functional exhaustion of CD4 + T cells induced by co‐stimulatory signals from myeloid leukaemia cells

doi: 10.1111/imm.12665

Figure Lengend Snippet: Up‐regulation of surface molecules related to activation and/or exhaustion on CD4 + T cells co‐cultured with different myeloid leukaemia cell lines for 96 hr under continuous stimuli conditions

Article Snippet: The monoclonal antibodies anti‐human‐CD4 (SK3), ‐CD3 (HIT3a), ‐CD69 (FN50), ‐CD25 (M‐A251), ‐CD14 (M5E2), ‐CD13 (L138), ‐CD274 (PD‐L1; MIH1) (Becton Dickinson, San Jose, CA); ‐LAG3 (FAB2319F) (R&D, Minneapolis, MN); ‐CD154 (24–31), ‐CD127 (hIL‐7r‐M21), ‐CD80 (2D10), ‐CD86 (IT2.2), ‐CD152 (CTLA‐4; L3D10), ‐CD275 (ICOS‐LG; 9F.8A4), ‐CD278 (ICOS; C398.4A), ‐CD279 (PD‐1; EH12.2H7), ‐FoxP3 (236A/E7), ‐TIM‐3 (F38‐2E2) (Biolegend, San Diego, CA) were used in immunophenotyping analyses.

Techniques: Activation Assay, Expressing

Journal: Immunology

Article Title: Functional exhaustion of CD4 + T cells induced by co‐stimulatory signals from myeloid leukaemia cells

doi: 10.1111/imm.12665

Figure Lengend Snippet: Comparative analysis of activation/exhaustion in Th cells co‐cultured with myeloid leukaemia cells or monocytes. (a) Percentage of Th cells carrying CD25, CD127, inducible T‐cell co‐stimulator (ICOS), CD69, T‐cell immunoglobulin and mucin domain‐containing protein 3 (TIM‐3), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD‐1) and cytotoxic T‐lymphocyte antigen 4 (CTLA‐4) were determined following continuous‐stimuli co‐culturing with representative myeloid leukaemia cell lines HL‐60 or THP‐1, or with CD14+ monocytes isolated from peripheral blood. For the specific marker, the mean percentage of CD4+ T cells cultured alone under continuous‐stimuli conditions is indicated with the dashed line. (b) Representative flow cytometry dot plots of LAG3/TIM‐3 and CD25/CD127 staining and (c) representative CFSE assay proliferation histograms of T cells obtained from continuous‐stimuli co‐culture experiments can be seen. Mean percentage ± SD values are given on the corresponding gates. (d) The number of TIM‐3+ LAG3+ T cells at the initiation (0 hr) and end (96 hr) of the co‐cultures are given. (e) interleukin‐2 (IL‐2), tumour necrosis factor‐α (TNF‐α) interferon‐γ and (IFN‐γ) levels were determined in the co‐culture supernatants with ELISA. (n ≥ 3, *P < 0·05, **P < 0·01). Mono., CD14+ monocytes; nd, not detected.

Article Snippet: The monoclonal antibodies anti‐human‐CD4 (SK3), ‐CD3 (HIT3a), ‐CD69 (FN50), ‐CD25 (M‐A251), ‐CD14 (M5E2), ‐CD13 (L138), ‐CD274 (PD‐L1; MIH1) (Becton Dickinson, San Jose, CA); ‐LAG3 (FAB2319F) (R&D, Minneapolis, MN); ‐CD154 (24–31), ‐CD127 (hIL‐7r‐M21), ‐CD80 (2D10), ‐CD86 (IT2.2), ‐CD152 (CTLA‐4; L3D10), ‐CD275 (ICOS‐LG; 9F.8A4), ‐CD278 (ICOS; C398.4A), ‐CD279 (PD‐1; EH12.2H7), ‐FoxP3 (236A/E7), ‐TIM‐3 (F38‐2E2) (Biolegend, San Diego, CA) were used in immunophenotyping analyses.

Techniques: Activation Assay, Cell Culture, Isolation, Marker, Flow Cytometry, Staining, CFSE Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: Functional exhaustion of CD4 + T cells induced by co‐stimulatory signals from myeloid leukaemia cells

doi: 10.1111/imm.12665

Figure Lengend Snippet: Effect of leukaemia‐derived co‐stimulatory signals on T helper (Th) cell exhaustion under continuous stimuli. Continuous‐stimuli co‐cultures of CD4+ T cells and myeloid leukaemia cells were performed in the presence of recombinant CTLA‐4‐Fc, inducible T‐cell co‐stimulator (ICOS)‐Fc, programmed cell death 1 (PD‐1)‐Fc or control mouse IgG (Iso. IgG). Together with aCD3 and culture media, these agents were also refreshed daily. At the end of 96 hr‐co‐culturing, the percentages of Th cells expressing lymphocyte activation gene 3 (LAG3)/ T‐cell immunoglobulin and mucin domain‐containing protein 3 (TIM‐3) exhaustion markers were determined by flow cytometry. (a) Representative contour plots of LAG3/TIM‐3 staining in CD4+ T cells co‐cultured with THP‐1 cells are given out of three independent experiments. Mean percentage ± SD values are given on the corresponding gates. (b) Change in the amount of T cells carrying exhaustion markers were calculated in comparison with the results obtained from the control co‐cultures established in the presence of Iso. IgG. (c) The results of co‐cultures established in the presence of both CTLA‐4‐Fc and ICOS‐Fc can be seen. A typical contour plot of LAG3/TIM‐3 expression and a bar graph showing the change in the amount of T cells expressing LAG3 and/or TIM‐3 is given. (d) Representative results from the continuous‐stimuli co‐cultures between Th cells and HL‐60 or THP‐1 cells enriched into CD86− or CD86+ sub‐populations [0·25 : 1 leukaemia cell : T‐cell ratio, 6·25 ng/ml (for HL‐60 co‐cultures), 25 ng/ml (for THP‐1 co‐cultures) aCD3]. (e) The percentage of CD4+ T cells expressing LAG3 and/or TIM3 in the continuous‐stimuli co‐cultures established with peripheral blood monocytes with or without aCD28 stimulation (2 : 1 leukaemia cell : T‐cell ratio, 25 ng/ml aCD3, 2 μg/ml aCD28), (n = 3, *P < 0·05).

Article Snippet: The monoclonal antibodies anti‐human‐CD4 (SK3), ‐CD3 (HIT3a), ‐CD69 (FN50), ‐CD25 (M‐A251), ‐CD14 (M5E2), ‐CD13 (L138), ‐CD274 (PD‐L1; MIH1) (Becton Dickinson, San Jose, CA); ‐LAG3 (FAB2319F) (R&D, Minneapolis, MN); ‐CD154 (24–31), ‐CD127 (hIL‐7r‐M21), ‐CD80 (2D10), ‐CD86 (IT2.2), ‐CD152 (CTLA‐4; L3D10), ‐CD275 (ICOS‐LG; 9F.8A4), ‐CD278 (ICOS; C398.4A), ‐CD279 (PD‐1; EH12.2H7), ‐FoxP3 (236A/E7), ‐TIM‐3 (F38‐2E2) (Biolegend, San Diego, CA) were used in immunophenotyping analyses.

Techniques: Derivative Assay, Recombinant, Expressing, Activation Assay, Flow Cytometry, Staining, Cell Culture

Journal: Immunology

Article Title: Functional exhaustion of CD4 + T cells induced by co‐stimulatory signals from myeloid leukaemia cells

doi: 10.1111/imm.12665

Figure Lengend Snippet: Lymphocyte activation gene 3 (LAG3), T‐cell immunoglobulin and mucin domain‐containing protein 3 (TIM‐3) and programmed cell death‐1 (PD‐1) expression on T helper (Th) cells in the bone marrow of patients with myeloproliferative disease. (a) The percentages of CD4+ T cells positive for LAG3, TIM‐3 and PD‐1 and of CD33+ myeloid blasts positive for CD86 and inducible T‐cell co‐stimulator ligand (ICOS‐LG) are plotted for each patient. The short lines indicate mean percentage values for particular markers, [Empty circles, acute myeloid leukaemia (AML) patients; filled circles, myelodysplastic syndrome (MDS) patients]. (b) Flow cytometry histograms are given for representative patient samples with distinct expression levels of LAG3, TIM‐3 or PD‐1 on gated CD4+ T cells. (c) Co‐expression of TIM‐3 and LAG3 on AML or MDS patient‐derived Th cells, (*P < 0·05). Representative flow cytometry dot‐plots of two patients showing low and high percentages of TIM‐3+ LAG3+ Th cells are shown on the right.

Article Snippet: The monoclonal antibodies anti‐human‐CD4 (SK3), ‐CD3 (HIT3a), ‐CD69 (FN50), ‐CD25 (M‐A251), ‐CD14 (M5E2), ‐CD13 (L138), ‐CD274 (PD‐L1; MIH1) (Becton Dickinson, San Jose, CA); ‐LAG3 (FAB2319F) (R&D, Minneapolis, MN); ‐CD154 (24–31), ‐CD127 (hIL‐7r‐M21), ‐CD80 (2D10), ‐CD86 (IT2.2), ‐CD152 (CTLA‐4; L3D10), ‐CD275 (ICOS‐LG; 9F.8A4), ‐CD278 (ICOS; C398.4A), ‐CD279 (PD‐1; EH12.2H7), ‐FoxP3 (236A/E7), ‐TIM‐3 (F38‐2E2) (Biolegend, San Diego, CA) were used in immunophenotyping analyses.

Techniques: Activation Assay, Expressing, Flow Cytometry, Derivative Assay