Journal: International Journal of Molecular Sciences

Article Title: High Pretransplant BAFF Levels and B-cell Subset Polarized towards a Memory Phenotype as Predictive Biomarkers for Antibody-Mediated Rejection

doi: 10.3390/ijms21030779

Figure Lengend Snippet: ( a ). Gating strategy of B cell subpopulations. Lymphocytes were identified by FSC (forward scatter) and SSC (side scatter). From lymphocyte population, CD19 + cells were selected, and based on them and according to IgD and CD27 markers, naïve (IgD + CD27 - ), unswitched memory (IgD + CD27 + ), and switched memory (IgD - CD27 + ) B cells were identified. Furthermore, based on CD19 + cells and according to IgD and CD38 markers, Bm1 (IgD + CD38 - ), Bm2 (IgD + CD38 + ), Bm2’ (IgD + CD38 high ), Bm3Bm4 (IgD - CD38 high ), eBm5 (IgD - CD38 + ), and Bm5 (IgD - CD38 - ) cells were identified. Transitional type 2 cells were selected using a different gating strategy in order to obtain a pure population. For this gating strategy, CD19 + lymphocytes were selected, and from them those cells CD10 + , CD20 + , CD21 + , CD5 - , CD24 + , and CD38 + . ( b ). Gating strategy of CD4 + and CD8 + T cell subpopulations. Lymphocytes were identified by FSC (forward scatter) and SSC (side scatter). From lymphocyte population, CD3 + cells were selected, and based on them, CD4 + and CD8 + cells. Since CD4 + and CD8 + cells, and according to CD62L and CD45RO markers, naïve (CD62L + CD45RO - ), central memory (CD62L + CD45RO + ), effector memory (CD62L - CD45RO + ), and TEMRA (CD62L - CD45RO - ) T cells were identified.

Article Snippet: The following monoclonal antibodies were used to T cell subpopulations identification: anti-CD62L-FITC clone DREG56 (Beckman Coulter, Brea, CA, USA), CD45RO-PE clone UCHL1 (BD Biosciences, San Diego, CA), CD28-PC5.5 clone L293, CD27-PE Cy7 Vio770 clone 1A4CD27, CCR7-APC clone REA108 (Miltenyi Biotec, Bergisch Gladbach, Germany), CD4-APC Vio770 clone VIT4, and CD3-VioBlue clone UCHT1 (Immunostep, Salamanca, Spain).

Techniques:

Journal: Nature Communications

Article Title: Metabolic requirements of NK cells during the acute response against retroviral infection

doi: 10.1038/s41467-021-25715-z

Figure Lengend Snippet: C57BL/6 mice were infected with FV for 3, 7, 12 and 28 days. Single-cell suspensions from spleens and bone marrow (BM) were prepared and used for the analysis of viral loads via Infectious Center assay ( a ). At least six mice from at least two individual experiments were used for the analysis. b Single-cell suspensions were stained for NK cell markers (CD3 − NK1.1 + CD49b + ) and analysed for the activation by measuring the early activation marker CD69. Naive mice were used as control. Mean values ± SEM were indicated by circles (bone marrow) and rectangles (spleen). Statistically significant differences between bone marrow and spleen ( a ) and CD69 + NK cells ( b ) were analysed by Mann–Whitney test. At 7 dpi, splenocytes were stained for NK cell markers and CD27 and CD11b ( c ). The effector phenotype of splenic NK cells ( e ) and NK cells from the bone marrow ( d ), KI-67, FasL, TNF, IFNγ and GzmB is displayed as spider plots. Data of NK cells from naive mice were displayed in blue and from FV infection in red. Statistically significant differences were analysed between naive and FV groups with an unpaired t -test (CD27 CD11b, KI-67, FasL, TNF (%), IFNγ (%)) or Mann–Whitney test (TNF (MFI), IFNγ (MFI)) within the bone marrow or spleens. A minimum of six mice from two independent experiments was used for the analysis. Significances are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. Applied statistical tests were two-sided. Source data are provided as a Source Data file. ns not significant.

Article Snippet: Antibodies used as follows: CD3 (17A2, FITC, 1:200, 100204, BioLegend), CD11b (M1/70, PE Cy7, 1:400, 101216, BioLegend), CD27 (LG.3A10, PE, 1:200, 558754, BD Pharmingen), CD49b (DX5, APC-Vio 770, 1:200, 130-105-249, Miltenyi Biotech), CD69 (H1.2F3, PerCP-Cy5.5, 1:200, 561931, BD Pharmingen), CD71 (R17217, APC, 1:200, 17-0711-82, eBioscience), CD98 (RL388, PE, 1:200, 12-0981-81, eBioscience), cMyc (D84C12, PE, 1:100, 14819, Cell Signaling), FasL (MFL3, PerCP-eFluor 710, 1:200, 46-5911-82, eBioscience), GzmB (NGZB, PE Cy7, 1:200, 25-8898-82, eBioscience), IFNγ (XMG1.2, APC, 1:100, 554413 BD Pharmingen), KI-67 (REA183, PE-Vio770, 1:200, 130-120-419, Miltenyi Biotech), NK1.1 (PK136, BV421, 1:200, 108732, BioLegend), Ter119 (TER-119, BV510, 1:200, 116237, BioLegend), TNF (MP6-XT22, PE Cy7, 1:100, 25-7321-82, eBioscience).

Techniques: Infection, Staining, Activation Assay, Marker, Control, MANN-WHITNEY

Journal: Nature Communications

Article Title: Metabolic requirements of NK cells during the acute response against retroviral infection

doi: 10.1038/s41467-021-25715-z

Figure Lengend Snippet: Splenic and bone marrow (BM) NK cells from naive mice or mice FV-infected for 7 days were analysed for cell size by analysing the FSC on NK cells. NK cells were gated on lymphocytes, single cells, viable, CD3 − , NK1.1 + CD49b + cells. A representative histogram of the FSC on NK cells from both organs are shown in a and displayed as a bar graph ± SEM in b . Statistically significant differences were analysed between naive and FV groups by a two-tailed unpaired t -test within the bone marrow or spleens. Experiments were repeated independently twice with similar results. The correlation between activated NK cells and NK cell size (FSC-A) was analysed and displayed in c . NK cells from naive mice are displayed in open circles whereas NK cells from FV-infected mice are displayed in grey circles. At least six mice per group from two independent experiments were used. FSC high and FSC low NK cells were analysed for the expression of CD98 and CD71 ( d ). NK1.1 expression was analysed on FSC high and FSC low NK cell population ( d , right-hand side). Experiments were repeated independently twice with similar results. Differences between FSC high and FSC low NK cells regarding CD71 and CD98 are shown in a bar graph in e . At least 14 mice from four independent experiments were used for the analysis. Statistically significant differences between FSC high and FSC low NK cells were analysed with a two-tailed Mann–Whitney test within the bone marrow or spleens. Data are presented as mean values ± SEM. Representative histogram of cMyc of NK cells in the spleen and bone marrow are shown in f. Experiments were repeated independently twice with similar results. The MFI of cMyc on NK cells ( g ) is displayed as bar graphs ± SEM and were analysed by a two-tailed unpaired t -test. At least six animals from two independent experiments were used for the analysis. cMyc + and cMyc − NK cells were further analysed for the cell size by measuring the FSC ( h ) and for proliferation by detecting KI-67 ( i ). At least six mice per group from two independent experiments were used. Data are presented as mean values ± SEM. Statistically significant differences in bone marrow or spleen were analysed with a two-sided unpaired t -test and displayed as * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Antibodies used as follows: CD3 (17A2, FITC, 1:200, 100204, BioLegend), CD11b (M1/70, PE Cy7, 1:400, 101216, BioLegend), CD27 (LG.3A10, PE, 1:200, 558754, BD Pharmingen), CD49b (DX5, APC-Vio 770, 1:200, 130-105-249, Miltenyi Biotech), CD69 (H1.2F3, PerCP-Cy5.5, 1:200, 561931, BD Pharmingen), CD71 (R17217, APC, 1:200, 17-0711-82, eBioscience), CD98 (RL388, PE, 1:200, 12-0981-81, eBioscience), cMyc (D84C12, PE, 1:100, 14819, Cell Signaling), FasL (MFL3, PerCP-eFluor 710, 1:200, 46-5911-82, eBioscience), GzmB (NGZB, PE Cy7, 1:200, 25-8898-82, eBioscience), IFNγ (XMG1.2, APC, 1:100, 554413 BD Pharmingen), KI-67 (REA183, PE-Vio770, 1:200, 130-120-419, Miltenyi Biotech), NK1.1 (PK136, BV421, 1:200, 108732, BioLegend), Ter119 (TER-119, BV510, 1:200, 116237, BioLegend), TNF (MP6-XT22, PE Cy7, 1:100, 25-7321-82, eBioscience).

Techniques: Infection, Two Tailed Test, Expressing, MANN-WHITNEY

Journal: Nature Communications

Article Title: Metabolic requirements of NK cells during the acute response against retroviral infection

doi: 10.1038/s41467-021-25715-z

Figure Lengend Snippet: Spleen, bone marrow (BM, tibia and femur of the right hind leg), lymph nodes (axillary, brachial, inguinal) and blood were collected from mice with loxP sites flanking exon 2 of the Slc7a5 gene crossed with transgenic mice expressing cre recombinase under the control of the NCR1 promoter (Slc7a5 NK-WT and Slc7a5 NK-KO mice). Single-cell suspensions were counted, stained for NK cell markers (CD3 − CD19 − NK1.1 + NKp46 + ) and measured by flow cytometry ( a ). Cell numbers in the blood were calculated per ml of blood. A minimum of three mice from two independent experiments were used and analysed by Mann–Whitney test. Data are presented as mean values ± SEM. Frequencies of CD27 and CD11b NK cell subsets of Slc7a5 NK-WT and Slc7a5 NK-KO mice are displayed in b for bone marrow and spleen. Data are presented as mean values ± SEM. NK cell kynurenine (kyn) uptake of unstimulated or IL-2/12-stimulated NK cells is displayed in c . Slc7a5 NK-WT and Slc7a5 NK-KO were infected with FV and at 7 dpi, the spleen and bone marrow of FV-infected Slc7a5 NK-WT , Slc7a5 NK-KO , or naive mice were harvested. A representative histogram from splenic NK cells (CD3 − CD49b + NK1.1 + ) from Slc7a5NK-WT and Slc7a5NK-KO mice is shown in d . Experiments were repeated independently twice with similar results. The absolute cell numbers of NK cells ( e ) and their proliferation ( f ) are displayed as bar graphs ± SEM. YAC-1 cells (right-hand side) or FV-induced tumour cells (FBL-3 cells, left-hand side) were stained with Tag-it-violet and co-incubated with isolated, splenic NK cells for 18 h ( g ). Viral loads were analysed by an IC assay in Slc7a5 NK-WT and Slc7a5 NK-KO mice ( h ). Data are presented as mean values ± SEM ( g , h ). A minimum of six mice per group from two independent experiments was used ( e – h ) and analysed by ordinary one-way ANOVA ( b , e , f ), unpaired t -test ( h ) or Kruskal–Wallis test ( g ). Significances are indicated as follows: ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file. BCH 2-Amino-2-norbornanecarboxylic acid, ns not significant.

Article Snippet: Antibodies used as follows: CD3 (17A2, FITC, 1:200, 100204, BioLegend), CD11b (M1/70, PE Cy7, 1:400, 101216, BioLegend), CD27 (LG.3A10, PE, 1:200, 558754, BD Pharmingen), CD49b (DX5, APC-Vio 770, 1:200, 130-105-249, Miltenyi Biotech), CD69 (H1.2F3, PerCP-Cy5.5, 1:200, 561931, BD Pharmingen), CD71 (R17217, APC, 1:200, 17-0711-82, eBioscience), CD98 (RL388, PE, 1:200, 12-0981-81, eBioscience), cMyc (D84C12, PE, 1:100, 14819, Cell Signaling), FasL (MFL3, PerCP-eFluor 710, 1:200, 46-5911-82, eBioscience), GzmB (NGZB, PE Cy7, 1:200, 25-8898-82, eBioscience), IFNγ (XMG1.2, APC, 1:100, 554413 BD Pharmingen), KI-67 (REA183, PE-Vio770, 1:200, 130-120-419, Miltenyi Biotech), NK1.1 (PK136, BV421, 1:200, 108732, BioLegend), Ter119 (TER-119, BV510, 1:200, 116237, BioLegend), TNF (MP6-XT22, PE Cy7, 1:100, 25-7321-82, eBioscience).

Techniques: Transgenic Assay, Expressing, Control, Staining, Flow Cytometry, MANN-WHITNEY, Infection, Incubation, Isolation

Journal: bioRxiv

Article Title: Highly differentiated CD4 T cells Unequivocally Identify Primary Resistance and Risk of Hyperprogression to PD-L1/PD-1 Immune Checkpoint Blockade in Lung Cancer

doi: 10.1101/320176

Figure Lengend Snippet: (A) Flow cytometry density plots of CD4 (upper graphs) and CD8 T cells (lower graphs) from young (left graphs) and senior (right graphs) healthy donors, according to CD28-CD27 expression profiles. Dashed lines within the upper left graph show the gates used to quantify poorly differentiated (CD28+ CD27+), intermediately differentiated (CD28+ CD27−) and T HD cells (CD28− CD27−). T HD cells are highlighted in each graph by a square. Percentages of each cell subset are indicated within the graphs. (B) Circulating highly differentiated CD4 / CD8 (upper graphs), and poorly differentiated CD4 / CD8 (lower graphs) subsets in age-matched healthy donors or NSCLC patients before undergoing immunotherapies. G1 and G2, groups of patients classified according to high T HD cells (G1) and low T HD cells (G2). N, number of patients used for analyses. Relevant statistical comparisons are shown by the U of Mann-Whitney test. (C) Flow cytometry density graphs of CD4 T HD from NSCLC G1 patients (upper graphs) or G2 patients (lower graphs) according to CD62L-CD45RA expression profiles. Dotted lines separate quadrants according to naïve/stem memory, central memory (CM), effector memory (EM) and effector phenotypes (EF), which include the percentage of cells in each quadrant. (D) As in (C) but representing data as scatter plot graphs for each patient classified according to G1 or G2 patient groups as indicated. Statistical comparisons performed by the U of Mann-Whitney. (E) Flow cytometry density plots of circulating CD4 T cells in G1 patients (upper left graph) and G2 patients (lower upper right graph) according to CD28-PD-1 expression profiles. The percentage of CD28+ PD-1+ CD4 T cells is indicated. The lower flow cytometry density graphs represent PD-1 and LAG3 up-regulation in CD4 T cells from a healthy donor (left graph) or an NSCLC patient (right graph) after T cell receptor (TCR) activation by A549 cells expressing a membrane bound anti-CD3 single-chain antibody. Percentages of cells within each quadrant are indicated. (F) Scatter plots representing the up-regulation of PD-1 after TCR activation as in (E) in healthy donors and NSCLC patients, separated into CD27+ and CD27-CD4 T cells. Relevant statistical comparisons are indicated, by the U of Mann Whitney. *** represents highly significant differences, respectively.

Article Snippet: The following fluorochrome-conjugated antibodies were used at the indicated dilutions: CD4-FITC (clone M-T466, reference 130–080–501, Miltenyi Biotec), CD4-APC-Vio770 (clone M-T466, reference 130–100–455, Miltenyi Biotec), CD4-PECy7 (clone SK3, reference 4129769, BD Biosciences,) CD27-APC (clone M-T271, reference 130–097–922, Miltenyi Biotec), CD27-PE (clone M-T271, reference 130–093–185, Miltenyi Biotec), CD45RA-FITC (reference 130–098–183, Miltenyi Biotec), CD62L-APC (reference 130–099–252, Miltenyi Biotech), CD28-PECy7 (clone CD28.2, reference 302926, Biolegend), PD-1-PE (clone EH12.2H7, reference 339905, Biolegend), CD8-FITC (clone SDK1, reference 344703, Biolegend), CD8-APC-Cy7(clone RFT-8, reference A15448, Molecular probes by Life technologies).

Techniques: Flow Cytometry, Expressing, MANN-WHITNEY, Activation Assay, Membrane

Journal: bioRxiv

Article Title: Highly differentiated CD4 T cells Unequivocally Identify Primary Resistance and Risk of Hyperprogression to PD-L1/PD-1 Immune Checkpoint Blockade in Lung Cancer

doi: 10.1101/320176

Figure Lengend Snippet: (A) Percentage of circulating CD4 T HD cells in treated patients along therapy from baseline (arrow, time 0). In green, patients with objective responses. In red, non-responders. Dotted line, the lowest discriminating cut-off value (40%) separating G1 from G2 patients. No responders were observed below this cut-off value in the cohort study. Below the graph, correlation of responses to T HD baseline values by the Fisher’s exact test. (B) Same as (A), but representing CD4 T PD (CD28+ CD27+) cells.

Article Snippet: The following fluorochrome-conjugated antibodies were used at the indicated dilutions: CD4-FITC (clone M-T466, reference 130–080–501, Miltenyi Biotec), CD4-APC-Vio770 (clone M-T466, reference 130–100–455, Miltenyi Biotec), CD4-PECy7 (clone SK3, reference 4129769, BD Biosciences,) CD27-APC (clone M-T271, reference 130–097–922, Miltenyi Biotec), CD27-PE (clone M-T271, reference 130–093–185, Miltenyi Biotec), CD45RA-FITC (reference 130–098–183, Miltenyi Biotec), CD62L-APC (reference 130–099–252, Miltenyi Biotech), CD28-PECy7 (clone CD28.2, reference 302926, Biolegend), PD-1-PE (clone EH12.2H7, reference 339905, Biolegend), CD8-FITC (clone SDK1, reference 344703, Biolegend), CD8-APC-Cy7(clone RFT-8, reference A15448, Molecular probes by Life technologies).

Techniques:

Journal: bioRxiv

Article Title: Highly differentiated CD4 T cells Unequivocally Identify Primary Resistance and Risk of Hyperprogression to PD-L1/PD-1 Immune Checkpoint Blockade in Lung Cancer

doi: 10.1101/320176

Figure Lengend Snippet: (A) Clinical case of a progressor with a G2 baseline profile associated to a “T HD burst” response. Flow cytometry density plots show baseline and post-first cycle CD4 (upper graphs) and CD8 T cells (lower graphs). Highly differentiated and poorly differentiated CD4 T cells are highlighted within doted square gates and hexagonal gates, respectively, together with their percentage. Below, CT scans of lung metastases and primary tumor progressing from baseline (left scans) after one month and a half (right scans) of therapy. Lesions are indicated with arrows. (B) Clinical case of a responder with a G1 baseline profile associated to systemic T HD reduction. CT scans show regression of hepatic and clavicular bone metastases (indicated with arrows) before (left scans) and after 3 months (right scans) of therapy. (C) Change in circulating CD4 T HD cells from baseline (pre-treatment) to post-first cycle of therapy. In red, progressors, in green objective responders. The 40% cut-off value separating G1 from G2 patients is shown. Below, increase and decrease in T HD cells within reponders or non-responders, as tested by paired t tests. (D) Waterfall plot of the relative changes in CD4 T HD cells in each patient from baseline to post-first cycle of therapy. Green bars, patients with objective responses; red bars, non-responders; purple, radiologically-confirmed hyperprogressors; black, suspected hyperprogressors. Below, correlation of T HD cell change with clinical outcome by the Fisher’s exact test.

Article Snippet: The following fluorochrome-conjugated antibodies were used at the indicated dilutions: CD4-FITC (clone M-T466, reference 130–080–501, Miltenyi Biotec), CD4-APC-Vio770 (clone M-T466, reference 130–100–455, Miltenyi Biotec), CD4-PECy7 (clone SK3, reference 4129769, BD Biosciences,) CD27-APC (clone M-T271, reference 130–097–922, Miltenyi Biotec), CD27-PE (clone M-T271, reference 130–093–185, Miltenyi Biotec), CD45RA-FITC (reference 130–098–183, Miltenyi Biotec), CD62L-APC (reference 130–099–252, Miltenyi Biotech), CD28-PECy7 (clone CD28.2, reference 302926, Biolegend), PD-1-PE (clone EH12.2H7, reference 339905, Biolegend), CD8-FITC (clone SDK1, reference 344703, Biolegend), CD8-APC-Cy7(clone RFT-8, reference A15448, Molecular probes by Life technologies).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Highly differentiated CD4 T cells Unequivocally Identify Primary Resistance and Risk of Hyperprogression to PD-L1/PD-1 Immune Checkpoint Blockade in Lung Cancer

doi: 10.1101/320176

Figure Lengend Snippet: (A) Flow cytometry density plots of Ki67 expression in T HD cells from a progressor (upper two graphs) and a responder (lower two graphs) at baseline and post-first cycle of therapy as indicated. Percentage and Ki67 mean fluorescent intensities in proliferating T HD cells are indicated within the graphs. (B) Change in Ki67 expression in CD4 T HD cells from responders and non-responders, as indicated. Only data was plotted from patients in which baseline and first-cycle Ki67 values were available. Paired t-tests were performed to compare the change from baseline to post-first cycle of therapy. (C) Dot plots of Ki67 expression in CD28+ CD27+ CD4 T cells (right graph) in non-responders and responders as indicated, in our cohort study. Differences were tested by the U of Mann-Whitney test. (D) Dot plots of changes in the percentage of circulating T HD differentiation subsets (as indicated) from baseline to post-first cycle of therapy, in patients exhibiting T HD bursts compared to responders. Data from patients with available CD62L-CD45RA profiles were used in the analyses. Relevant statistical comparisons are shown within the graphs, using paired t tests; N, number of patients used in the analyses; * indicate significant differences.

Article Snippet: The following fluorochrome-conjugated antibodies were used at the indicated dilutions: CD4-FITC (clone M-T466, reference 130–080–501, Miltenyi Biotec), CD4-APC-Vio770 (clone M-T466, reference 130–100–455, Miltenyi Biotec), CD4-PECy7 (clone SK3, reference 4129769, BD Biosciences,) CD27-APC (clone M-T271, reference 130–097–922, Miltenyi Biotec), CD27-PE (clone M-T271, reference 130–093–185, Miltenyi Biotec), CD45RA-FITC (reference 130–098–183, Miltenyi Biotec), CD62L-APC (reference 130–099–252, Miltenyi Biotech), CD28-PECy7 (clone CD28.2, reference 302926, Biolegend), PD-1-PE (clone EH12.2H7, reference 339905, Biolegend), CD8-FITC (clone SDK1, reference 344703, Biolegend), CD8-APC-Cy7(clone RFT-8, reference A15448, Molecular probes by Life technologies).

Techniques: Flow Cytometry, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Highly differentiated CD4 T cells Unequivocally Identify Primary Resistance and Risk of Hyperprogression to PD-L1/PD-1 Immune Checkpoint Blockade in Lung Cancer

doi: 10.1101/320176

Figure Lengend Snippet: (A) Spider plot of change in target lesions. Red, patients that started therapy with a negative baseline profile. These patients presented progressive disease, or growth of lesions. (B) Spider plot of change in target lesions of progressors before and after the start of immunotherapy. (C) Scatter plot of baseline T HD cell values in hyperprogressors, suspected hyperprogressors and progressors, as indicated. Dotted line shows the 40% cut-off value separating G1 from G2 patients. Below, correlation of baseline T HD cells with radiologically-confirmed hyperprogressors by a Fisher’s exact test. Suspected hyperprogressors were excluded. (D) Scatter plot of changes in CD4 T HD percentage from baseline to post-first cycle of therapy in radiologically-confirmed hyperprogressors, suspected hyperprogressors and progressors. Dotted line separates T HD increases from decreases. Differences were tested by U of Mann-Whitney test. Suspected hyperprogressors were excluded. Below, correlation of T HD burst with radiologically-confirmed hyperprogressors by a Fisher’s exact test. N, number of patients in each group; Comparisons of CD4 T HD cells and changes in CD4 T HD cells were performed with the U of Mann-Whitney excluding suspected hyperprogressors; N, number of patients used in the analyses; *,**, ** indicates significant, very and highly significant differences.

Article Snippet: The following fluorochrome-conjugated antibodies were used at the indicated dilutions: CD4-FITC (clone M-T466, reference 130–080–501, Miltenyi Biotec), CD4-APC-Vio770 (clone M-T466, reference 130–100–455, Miltenyi Biotec), CD4-PECy7 (clone SK3, reference 4129769, BD Biosciences,) CD27-APC (clone M-T271, reference 130–097–922, Miltenyi Biotec), CD27-PE (clone M-T271, reference 130–093–185, Miltenyi Biotec), CD45RA-FITC (reference 130–098–183, Miltenyi Biotec), CD62L-APC (reference 130–099–252, Miltenyi Biotech), CD28-PECy7 (clone CD28.2, reference 302926, Biolegend), PD-1-PE (clone EH12.2H7, reference 339905, Biolegend), CD8-FITC (clone SDK1, reference 344703, Biolegend), CD8-APC-Cy7(clone RFT-8, reference A15448, Molecular probes by Life technologies).

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Highly differentiated CD4 T cells Unequivocally Identify Primary Resistance and Risk of Hyperprogression to PD-L1/PD-1 Immune Checkpoint Blockade in Lung Cancer

doi: 10.1101/320176

Figure Lengend Snippet: (A) Kaplan-Meier plot for PFS in patients undergoing immune checkpoint inhibitor therapies stratified by strict baseline negative and positive T cell profiles as defined in the text. Patients starting therapy with a negative baseline profile had an overall response rate (ORR) of 0% and all experienced progression or death by week 9. ORR was 38.9% for patients with a positive baseline profile, and the 12-week PFS was 44%. (B) As in (A) but stratifying patients according to hyperprogression assessed by immunological criteria (negative CD4 T HD baseline profile-significant T HD burst). Patients classified as immunological progressors progressed or died by week 7. (C) Kaplan-Meier plot of time of diagnosis to enrolment in patients stratified by positive or negative CD4 T HD profiles as indicated, demonstrating no significant prognostic value. (D) ROC analysis of baseline CD4 T HD quantification as a predictive biomarker. Within the graph, highest cut-off value of CD4 T HD cells to discriminate intrinsic responders with 100% specificity. **, indicates very significant differences. (E) Scatter plots of percentages of baseline T HD differentiation subsets as indicated on top of each graph in responders and non-responders from G1 patients. Statistical comparisons were performed with the U of Mann-Whitney test. Right bottom, correlation of the percentage of naïve/stem memory CD4 T HD cells with objective responses in G1 patients by a Fisher’s exact test. (F) Scatter plot of the percentage of baseline CD4 T HD cells according to tumor expression levels as shown in the legend.

Article Snippet: The following fluorochrome-conjugated antibodies were used at the indicated dilutions: CD4-FITC (clone M-T466, reference 130–080–501, Miltenyi Biotec), CD4-APC-Vio770 (clone M-T466, reference 130–100–455, Miltenyi Biotec), CD4-PECy7 (clone SK3, reference 4129769, BD Biosciences,) CD27-APC (clone M-T271, reference 130–097–922, Miltenyi Biotec), CD27-PE (clone M-T271, reference 130–093–185, Miltenyi Biotec), CD45RA-FITC (reference 130–098–183, Miltenyi Biotec), CD62L-APC (reference 130–099–252, Miltenyi Biotech), CD28-PECy7 (clone CD28.2, reference 302926, Biolegend), PD-1-PE (clone EH12.2H7, reference 339905, Biolegend), CD8-FITC (clone SDK1, reference 344703, Biolegend), CD8-APC-Cy7(clone RFT-8, reference A15448, Molecular probes by Life technologies).

Techniques: Biomarker Discovery, MANN-WHITNEY, Expressing