Journal:

Article Title: Depletion of CD8 + cells abolishes memory in acquired immunity against Chlamydia pneumoniae in BALB/c mice

doi: 10.1046/j.1365-2567.1999.00809.x

Figure Lengend Snippet: The numbers of cultured Chlamydia pneumoniae inclusion-forming units (IFUs) from the lungs of wild-type and in vivo CD8- or CD4-depleted BALB/c mice during primary infection and reinfection. Data represents the mean logarithmic values obtained from individual mice. The data for wild-type mice are the same as detailed in the legend to Figure 1. Numbers of mice used in the depletion experiments are shown in boxes inside the bars. The detection limit of the culture assay, 1·3 IFU/lung, is shown by an arrow. *Statistically significant difference compared with wild-type mice (P < 0·01, P < 0·01 after primary challenge and P = 0·001, P < 0·001, P < 0·05 after rechallenge). ND, not determined.

Article Snippet: The 6–8-week-old mice were depleted of a specific lymphocyte subset by 0·5 mg/dose of anti-CD8 (clone YTS 169, a kind gift from Dr R. L. Coffman, DNAX Research Institute, Palo Alto, CA) or anti-CD4 (clone GK 1·5, DNAX Research Institute) monoclonal antibodies (mAbs) that had been produced in serum-free media (by DiaBor Ltd, Oulu, Finland).

Techniques: Cell Culture, In Vivo, Infection

Journal:

Article Title: Depletion of CD8 + cells abolishes memory in acquired immunity against Chlamydia pneumoniae in BALB/c mice

doi: 10.1046/j.1365-2567.1999.00809.x

Figure Lengend Snippet: Induction of (a) proliferation and (b) secretion of tumour necrosis factor-α (TNF-α), (c) interferon-γ (IFN-γ) and (d) interleukin-10 (IL-10) by pulmonary cells, isolated on the indicated days after primary Chlamydia pneumoniae infection from wild-type, CD8-depleted and nude mice, in response to in vitro stimulation with inactivated C. pneumoniae. In the late stage of infection (days 18–33), data from typically two different time-points are combined. In panel (d) the scale is one-tenth that of panels (b) and (c). The proliferation index was calculated as described in the Materials and methods. Cytokine secretion results are expressed as C. pneumoniae-induced cytokine secretion after subtraction of the background secretion. BD, below the detection limit; ND, not determined.

Article Snippet: The 6–8-week-old mice were depleted of a specific lymphocyte subset by 0·5 mg/dose of anti-CD8 (clone YTS 169, a kind gift from Dr R. L. Coffman, DNAX Research Institute, Palo Alto, CA) or anti-CD4 (clone GK 1·5, DNAX Research Institute) monoclonal antibodies (mAbs) that had been produced in serum-free media (by DiaBor Ltd, Oulu, Finland).

Techniques: Isolation, Infection, In Vitro

Journal: Clinical and Translational Medicine

Article Title: A four‐factor immune risk score signature predicts the clinical outcome of patients with spinal chordoma

doi: 10.1002/ctm2.4

Figure Lengend Snippet: Representative immunofluorescence images showing tumor‐infiltrating lymphocytes level within tumor and stromal subregions of chordoma tissues. Scale bar = 50 μm. For multiplex immunofluorescence staining, 4‐μm thick tumor sections were dewaxed and rehydrated. After antigen retrieval and blocking, tissue sections were treated with primary cytokeratin 19 antibody (1:5000). HRP‐conjugated secondary antibodies were then incubated at room temperature for 1 hour followed by tyramide‐based HRP activation at 37°C for 20 min. This was repeated five more times using the following antibodies: CD3 (1:10), Foxp3 (1:500), CD8 (1:500), CD20 (1:5000), and PD‐1 (1:10000). Nuclei were stained with DAPI.

Article Snippet: One batch was treated with a mix of anti‐CD3 allophycocyanin (APC)‐CY7‐conjugated (0.1 mg/mL; BD Biosciences), Percp‐conjugated anti‐CD8 (0.05 mg/mL; BD Biosciences), anti‐CD20 fluorescein isothiocyanate conjugated (0.012 mg/mL; eBioscience), anti‐PD‐1 APC‐conjugated (0.1 mg/mL; BD Biosciences), and anti‐Foxp3 phycoerythrin‐conjugated antibodies (0.1 mg/mL; eBioscience), followed by fix and permeabilization buffer (eBioscience) for 30 minutes in the dark on ice.

Techniques: Immunofluorescence, Multiplex Assay, Staining, Blocking Assay, Incubation, Activation Assay

Journal: Clinical and Translational Medicine

Article Title: A four‐factor immune risk score signature predicts the clinical outcome of patients with spinal chordoma

doi: 10.1002/ctm2.4

Figure Lengend Snippet: Validation of quantitative immunofluorescence assay for measuring TILs levels by comparison to flow cytometry on fresh chordoma samples. A, Representative field of view showing TILs subpopulation proportions from quantitative immunofluorescence on chordoma tissue. B, Representative example of flow cytometry on chordoma tissue, in which numbers in this picture represent the percentage of TILs analyzed. The total lymphocytes (left) were gated into PD‐1 subpopulation, distinct CD3 and CD20 populations (middle), and overlapping CD3 and CD8, as well as CD3 and Foxp3 populations (right). C‐H, Percentage of lymphocytes and subpopulations was similar when analyzed by flow cytometry and quantitative immunofluorescence. For multiplex immunofluorescence staining, 4‐μm‐thick tumor sections were dewaxed and rehydrated. After antigen retrieval and blocking, tissue sections were treated with primary cytokeratin 19 antibody (1:5000). HRP‐conjugated secondary antibodies were then incubated at room temperature for 1 hour followed by tyramide‐based HRP activation at 37°C for 20 min. This was repeated five more times using the following antibodies: CD3 (1:10), Foxp3 (1:500), CD8 (1:500), CD20 (1:5000), and PD‐1 (1:10000). Nuclei were stained with DAPI. For flow cytometry, single‐cell suspensions with a concentration of 10 7 cells/mL in phosphate‐buffered saline [PBS] (containing 5% fetal bovine serum [FBS] and 0.1% sodium azide) were split into five equal proportions of 10 6 cells. One proportion was treated with a mix of anti‐CD3 APC‐CY7‐conjugated (0.1 mg/mL), anti‐CD8 Percp‐conjugated (0.05 mg/mL), anti‐CD20 fluorescein isothiocyanate conjugated antibodies (0.012 mg/mL), anti‐PD‐1 APC‐conjugated antibodies (0.1 mg/mL), anti‐Foxp3 PE‐conjugated antibodies(0.1mg/mL), and fix and permeabilization buffer for 30 min in the dark on ice. The other four proportions were used as compensation controls. Flow cytometry consistently determined a greater proportion of CD3‐ and CD8‐positive lymphocytes, while quantitative immunofluorescence consistently resulted in a greater proportion of lymphocytes, B cells, as well as PD‐1 + and Foxp3 + T cells.

Article Snippet: One batch was treated with a mix of anti‐CD3 allophycocyanin (APC)‐CY7‐conjugated (0.1 mg/mL; BD Biosciences), Percp‐conjugated anti‐CD8 (0.05 mg/mL; BD Biosciences), anti‐CD20 fluorescein isothiocyanate conjugated (0.012 mg/mL; eBioscience), anti‐PD‐1 APC‐conjugated (0.1 mg/mL; BD Biosciences), and anti‐Foxp3 phycoerythrin‐conjugated antibodies (0.1 mg/mL; eBioscience), followed by fix and permeabilization buffer (eBioscience) for 30 minutes in the dark on ice.

Techniques: Immunofluorescence, Flow Cytometry, Multiplex Assay, Staining, Blocking Assay, Incubation, Activation Assay, Concentration Assay

Journal: Clinical and Translational Medicine

Article Title: A four‐factor immune risk score signature predicts the clinical outcome of patients with spinal chordoma

doi: 10.1002/ctm2.4

Figure Lengend Snippet: Distribution of IRS, recurrence status, and survival status among the training (A) and validation (B) chordoma samples. The heatmap (bottom) shows the profile of four immune features included for the IRS construction in chordoma patients. Columns represent patients who were sorted descendingly by their IRS levels. Red dotted line represents the IRS cutoff dividing patients into high and low subgroups. B, Receiver operating characteristics curves show the sensitivity and specificity for the IRS (as a continuous variable) in predicting LRFS and OS of patients from the training (C) and validation (D) cohort. Abbreviations: AUC, area under the curve; IRS, immune risk score; LRFS, local recurrence‐free survival; OS, overall survival; sCD8, stromal CD8 + TILs; sFoxp3, stromal Foxp3 + TILs; tFoxp3, tumor Foxp3 + TILs; TILs, tumor‐infiltrating lymphocytes; tPD‐1, tumor PD‐1 + TILs.

Article Snippet: One batch was treated with a mix of anti‐CD3 allophycocyanin (APC)‐CY7‐conjugated (0.1 mg/mL; BD Biosciences), Percp‐conjugated anti‐CD8 (0.05 mg/mL; BD Biosciences), anti‐CD20 fluorescein isothiocyanate conjugated (0.012 mg/mL; eBioscience), anti‐PD‐1 APC‐conjugated (0.1 mg/mL; BD Biosciences), and anti‐Foxp3 phycoerythrin‐conjugated antibodies (0.1 mg/mL; eBioscience), followed by fix and permeabilization buffer (eBioscience) for 30 minutes in the dark on ice.

Techniques: