cytomegalovirus pp65 protein  (Miltenyi Biotec)

Journal: Oncoimmunology

Article Title: Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selective eliminate carcinoma cells

doi: 10.1080/2162402X.2023.2207868

Figure Lengend Snippet: EpCAM-ReTARG pp65 selectively and dose-dependently binds to EpCAM pos cancer cells. (a) Schematic representation of transmembrane(TM)-ReTARG pp65 and EpCAM-ReTARG pp65 fusion proteins. The anti-EpCAM Fab domain of EpCAM-ReTARG pp65 was constructed using published VH and VL gene sequences of the humanized antibody fragment scFv 4D5 MOC-B . (b) Proposed mode-of-action of EpCAM-ReTARG pp65 . EpCAM-ReTARG pp65 binds via its anti-EpCAM Fab antibody domain to cell surface-exposed EpCAM on cancer cells. TPR peptide-equipped HLA-B × 07:02/β2 M recruits cognate anti‐CMV pp65 CD8 pos T cells, which mediate cancer cell death by releasing apoptosis-inducing agents, such as granzymes and perforins. (c) SDS-PAGE analysis with Coomassie brilliant blue staining of EpCAM-ReTARG pp65 under non-reducing (NR, lane 1) and reducing (R, lane 2) conditions. M = marker. (d) Concentration-dependent binding of EpCAM-ReTARG pp65 and Mock-ReTARG pp65 to EpCAM pos PC-3 M cancer cells. (e) Binding of EpCAM-ReTARG pp65 (0.5 μg/ml) to EpCAM pos PC-3 M vs PC-3 M.EpCAM-KO cancer cells. (f) Binding of EpCAM-ReTARG pp65 (1 μg/ml) to EpCAM neg A375M and A375 M.EpCAM melanoma cells. A375M.TM-ReTARG pp65 cells were used as a positive control. Graphs in D-F were assessed by flow cytometry and representative images with two technical replicates are shown (mean ± SD). n = 3. Statistical analysis in D was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test. Statistical analysis in F was performed using unpaired t-test (anti-HLA-B7-APC vs EpCAM-ReTARG pp65 per cell line). (ns = not significant, ***p < .001).

Article Snippet: PBMCs were stimulated with either recombinant CMV pp65 (Miltenyi Biotech) or T cell-activated IE-1 (Lophius Biosciences) protein solution for 2 d. Next, stimulated PBMCs were harvested, resuspended in fresh X-VIVO15 medium supplemented with 500 U/ml IL-2 (Immunotools), and cultured for an additional 4 d. Subsequently, CMV pp65 - and CMV IE-1 -stimulated PBMCs were cultured on a feeder layer of OvCAR-3.pp65 cells and OvCAR-3.IE-1 at an effector-to-target (E:T) cell ratio of 7.5:1.

Techniques: Construct, SDS Page, Staining, Marker, Concentration Assay, Binding Assay, Positive Control, Flow Cytometry

Journal: Oncoimmunology

Article Title: Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selective eliminate carcinoma cells

doi: 10.1080/2162402X.2023.2207868

Figure Lengend Snippet: Ex vivo-expanded anti‐CMV pp65 CD8 pos T cells potently eliminate CMV pp65‐expressing cancer cells at low E:T cell ratios (a) Cytotoxic capacity of PBMCs isolated freshly from 4 CMV‐seropositive/HLA‐B × 07:02 pos donors and 1 CMV-seronegative/HLA‐B × 07:02 pos donor toward OvCAR-3.Pp65 cancer cells (E:T cell ratio = 20:1). Elimination of OvCAR-3.Pp65 cancer cells was evaluated after 4 d using a standard LDH release cytotoxicity assay. (b) Percentage of anti‐CMV pp65 CD8 pos T cells per donor was evaluated by staining for TPR-dextramer pos T cells. (c) Cytotoxic capacity of stimulated and expanded anti‐CMV pp65 CD8 pos T cells toward OvCAR-3.Pp65 cells (E:T cell ratio = 2:1). Apoptotic cancer cell death was assessed using Annexin-V/PI staining after 24 h. (d) Antigen-specific activation status (CD137 exposure) of anti‐CMV pp65 CD8 pos T cells as used in C. (e) Cytotoxic capacity of stimulated and expanded anti‐CMV pp65 CD8 pos T cells toward OvCAR-3, OvCAR-3.Pp65 (Hla‐b × 07:02‐matched) and FaDu.Pp65 (Hla‐b × 07:02‐unmatched) cancer cells at increasing E:T cell ratios. Apoptotic cancer cell death was assessed using Annexin-V/PI staining after 24 h. OvCAR-3.transmembrane(TM)-ReTARG pp65 cells were used as a positive control. Graphs a and B show representative images with two technical replicates (mean ± SD). Graphs C and D: n = 1 (two technical replicates). Graph E: n = 3 (two technical replicates), mean ± SD are shown and statistical analysis was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test (OvCAR-3.Pp65 vs. FaDu.Pp65 cells). (*p < .05, ***p < .001).

Article Snippet: PBMCs were stimulated with either recombinant CMV pp65 (Miltenyi Biotech) or T cell-activated IE-1 (Lophius Biosciences) protein solution for 2 d. Next, stimulated PBMCs were harvested, resuspended in fresh X-VIVO15 medium supplemented with 500 U/ml IL-2 (Immunotools), and cultured for an additional 4 d. Subsequently, CMV pp65 - and CMV IE-1 -stimulated PBMCs were cultured on a feeder layer of OvCAR-3.pp65 cells and OvCAR-3.IE-1 at an effector-to-target (E:T) cell ratio of 7.5:1.

Techniques: Ex Vivo, Expressing, Isolation, Cytotoxicity Assay, Staining, Activation Assay, Positive Control

Journal: Oncoimmunology

Article Title: Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selective eliminate carcinoma cells

doi: 10.1080/2162402X.2023.2207868

Figure Lengend Snippet: EpCAM-ReTARG pp65 engages anti‐CMV pp65 CD8 pos T cells in an EpCAM-selective manner. Cytotoxic capacity of anti‐CMV pp65 CD8 pos T cells toward (a) EpCAM neg A375M, (b) A375 M.EpCAM, and (c) A375M.transmembrane(TM)-ReTARG pp65 melanoma cells at increasing E:T cell ratios in the presence (or absence) of EpCAM-ReTARG pp65 (100 ng/ml). Apoptotic cancer cell death was assessed using Annexin-V/PI staining after 24 h. A375M.TM-ReTARG pp65 cells were used as positive control. (d) Cytotoxic capacity of anti‐CMV pp65 CD8 pos T cells toward PC-3 M and PC-3 M.EpCAM-KO cancer cells at increasing E:T cell ratios in the presence (or absence) of EpCAM-ReTARG pp65 (100 ng/ml). Apoptotic cancer cell death was assessed using Annexin-V/PI staining after 24 h. (e,f) Cytotoxic capacity of anti‐CMV pp65 CD8 pos T cells toward PC-3 M and PC-3 M.EpCAM-KO cancer cells (E:T cell ratio = 4:1) in the presence (or absence) of EpCAM-ReTARG pp65 (100 ng/ml) using a conditionally fluorescent caspase-3/8 substrate. Representative images shown were taken after 15 h at 10× magnification. Quantification of green fluorescent cells over time is shown. The ex vivo-expanded T cells used were from CMV‐seropositive/HLA‐B × 07:02 pos donor #2. Graphs A-D: n = 3 (two technical replicates), mean ± SD are shown. Graph F shows a representative image with two technical replicates (mean ± SD). Statistical analysis in A-C was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test. Statistical analysis in D+F was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test (PC-3 M + EpCAM-ReTARG pp65 vs PC-3 M.EpCAM-KO + EpCAM-ReTARG pp65 ). (ns = not significant, *p < .05, **p < .01, ***p < .001).

Article Snippet: PBMCs were stimulated with either recombinant CMV pp65 (Miltenyi Biotech) or T cell-activated IE-1 (Lophius Biosciences) protein solution for 2 d. Next, stimulated PBMCs were harvested, resuspended in fresh X-VIVO15 medium supplemented with 500 U/ml IL-2 (Immunotools), and cultured for an additional 4 d. Subsequently, CMV pp65 - and CMV IE-1 -stimulated PBMCs were cultured on a feeder layer of OvCAR-3.pp65 cells and OvCAR-3.IE-1 at an effector-to-target (E:T) cell ratio of 7.5:1.

Techniques: Staining, Positive Control, Ex Vivo

Journal: Oncoimmunology

Article Title: Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selective eliminate carcinoma cells

doi: 10.1080/2162402X.2023.2207868

Figure Lengend Snippet: EpCAM-ReTARG pp65 selectively activates cytotoxic effector potential of anti‐CMV pp65 CD8 pos T cells. (a) Antigen-specific activation status (CD137 exposure) of anti‐CMV pp65 CD8 pos T cells in presence (or absence) of PC-3 M cancer cells (E:T cell ratio = 2:1), EpCAM-ReTARG pp65 and Mock-ReTARG pp65 (both 100 ng/ml) after 24 h. Subsequently, conditioned culture media was collected and analyzed for T cell-secreted Granzyme B and IFNγ by appropriate ELISAs. (b,c) Analysis of proliferation and subsequent aggregate formation of CFSE-labeled anti‐CMV pp65 CD8 pos effector T cells cocultured (or not) with PC-3 M target cells (E:T cell ratio = 2:1) in presence of EpCAM-ReTARG pp65 and Mock-ReTARG pp65 (both 100 ng/ml) by live cell imaging. Representative images shown were taken after 40 h at a 10× magnification. Size of T cell clusters (Red Object Area (μm 2 /image)) is shown as fold change over time. (d) Capacity of EpCAM-ReTARG pp65 and Mock-ReTARG pp65 at increasing concentrations to activate anti‐CMV pp65 CD8 pos T cells in the presence of EpCAM pos PC-3 M cells (E:T cell ratio = 2:1). Conditioned culture media was collected after 24 h and T cell-secreted IFNγ quantified by ELISA. (e) Capacity of EpCAM-ReTARG pp65 (100 ng/ml) to activate anti‐CMV pp65 CD8 pos T cells in the presence of EpCAM pos PC-3 M and PC-3 M.EpCAM-KO cancer cells at increasing E:T cell ratios. Conditioned culture media was collected after 24 h and T cell-secreted IFNγ quantified by ELISA. The ex vivo-expanded T cells used were from CMV‐seropositive/HLA‐B × 07:02 pos donor #2. All graphs show representative images with two technical replicates (mean ± SD). Statistical analysis in a was performed using unpaired t-test. Statistical analysis in D was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test. Statistical analysis in E was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test (PC-3 M + EpCAM-ReTARG pp65 vs PC-3 M.EpCAM-KO + EpCAM-ReTARG pp65 ). (*p < .05, **p < .01, ***p < .001).

Article Snippet: PBMCs were stimulated with either recombinant CMV pp65 (Miltenyi Biotech) or T cell-activated IE-1 (Lophius Biosciences) protein solution for 2 d. Next, stimulated PBMCs were harvested, resuspended in fresh X-VIVO15 medium supplemented with 500 U/ml IL-2 (Immunotools), and cultured for an additional 4 d. Subsequently, CMV pp65 - and CMV IE-1 -stimulated PBMCs were cultured on a feeder layer of OvCAR-3.pp65 cells and OvCAR-3.IE-1 at an effector-to-target (E:T) cell ratio of 7.5:1.

Techniques: Activation Assay, Labeling, Live Cell Imaging, Enzyme-linked Immunosorbent Assay, Ex Vivo

Journal: Oncoimmunology

Article Title: Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selective eliminate carcinoma cells

doi: 10.1080/2162402X.2023.2207868

Figure Lengend Snippet: EpCAM-ReTARG pp65 redirects the cytotoxic activity of anti‐CMV pp65 CD8 pos T cells toward EpCAM pos carcinoma cell lines of diverse origins and primary patient-derived cancer cells. (a) Cytotoxic capacity of anti‐CMV pp65 CD8 pos T cells toward PC-3 M cancer cells (E:T cell ratio = 2:1) in the presence of increasing concentrations of EpCAM-ReTARG pp65 and Mock-ReTARG pp65 . Cytotoxic capacity of anti‐CMV pp65 CD8 pos T cells toward (b) PC-3 M; (c) OvCAR-3; (d) NCI-H292 cancer cells at increasing E:T cell ratios in the presence (or absence) of EpCAM-ReTARG pp65 and Mock-ReTARG pp65 (both 100 ng/ml). (e) Cytotoxic capacity of anti‐CMV pp65 CD8 pos T cells toward cancer samples from 3 patients with stage IIIC high‐grade serous ovarian cancer at increasing E:T cell ratios in the presence (or absence) of EpCAM-ReTARG pp65 (100 ng/ml). Data obtained from all patient samples were pooled. (f) Cancer cell elimination at E:T cell ratio 5:1 is shown per patient as Δ(+EpCAM-ReTARG pp65 - anti-CMV pp65 T cells) in %. The ex vivo-expanded T cells used were from CMV‐seropositive/HLA‐B × 07:02 pos donor #2. Graphs A-D: n = 3 (two technical replicates), mean ± SD are shown. Graph E shows biological replicates (mean ± SD). Statistical analysis in A-D was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test (EpCAM-ReTARG pp65 vs Mock-ReTARG pp65 ). Statistical analysis in E was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test. (*p < .05, ***p < .001).

Article Snippet: PBMCs were stimulated with either recombinant CMV pp65 (Miltenyi Biotech) or T cell-activated IE-1 (Lophius Biosciences) protein solution for 2 d. Next, stimulated PBMCs were harvested, resuspended in fresh X-VIVO15 medium supplemented with 500 U/ml IL-2 (Immunotools), and cultured for an additional 4 d. Subsequently, CMV pp65 - and CMV IE-1 -stimulated PBMCs were cultured on a feeder layer of OvCAR-3.pp65 cells and OvCAR-3.IE-1 at an effector-to-target (E:T) cell ratio of 7.5:1.

Techniques: Activity Assay, Derivative Assay, Ex Vivo

Journal: Oncoimmunology

Article Title: Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selective eliminate carcinoma cells

doi: 10.1080/2162402X.2023.2207868

Figure Lengend Snippet: EpCAM-ReTARG pp65 redirects anti‐CMV pp65 CD8 pos T cells to potently eliminate cancer cells in absence of excessive cytokine release. (a) Cytotoxic capacity of anti‐CMV pp65 CD8 pos T cells toward EpCAM pos PC-3 M cancer cells (E:T cell ratio = 2:1) in the presence of increasing concentrations of EpCAM-ReTARG pp65 and BiTE solitomab. Apoptotic cancer cell death was assessed using Annexin-V/PI staining after 24 h. Background killing was subtracted and maximal cancer cell elimination set to 100%. EC 50 = half maximal effective concentration. Conditioned cell culture supernatants were collected and analyzed for T cell-secreted (b) IFNγ and (c) Granzyme B by ELISA. (d) Comparison of IFNγ and Granzyme B levels at relevant concentrations of EpCAM-ReTARG pp65 and solitomab, which convey comparable (maximal) T cell-mediated cancer cell elimination. Of note, BiTE solitomab acts by redirecting the cytotoxic activity of T cells toward EpCAM pos cancer cells by activating the CD3ε chain of their TCRs, irrespective of their intrinsic specificity. To allow for an appropriate comparison of EpCAM-ReTARG pp65 and solitomab with equal numbers of available effector T cells, the amount of ex vivo-expanded anti‐CMV pp65 CD8 pos T cells in the presence of solitomab was adjusted to match the percentage of TPR-dextramer pos cells, which was found to be 50%. The ex vivo-expanded T cells used were from CMV‐seropositive/HLA‐B × 07:02 pos donor #2. Graph A: n = 3 (two technical replicates), mean ± SD are shown. Graphs B-D show representative images with two technical replicates (mean ± SD). Statistical analysis in D was performed using unpaired t-test (*p < .05, **p < .01).

Article Snippet: PBMCs were stimulated with either recombinant CMV pp65 (Miltenyi Biotech) or T cell-activated IE-1 (Lophius Biosciences) protein solution for 2 d. Next, stimulated PBMCs were harvested, resuspended in fresh X-VIVO15 medium supplemented with 500 U/ml IL-2 (Immunotools), and cultured for an additional 4 d. Subsequently, CMV pp65 - and CMV IE-1 -stimulated PBMCs were cultured on a feeder layer of OvCAR-3.pp65 cells and OvCAR-3.IE-1 at an effector-to-target (E:T) cell ratio of 7.5:1.

Techniques: Staining, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison, Activity Assay, Ex Vivo

Journal: Oncoimmunology

Article Title: Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selective eliminate carcinoma cells

doi: 10.1080/2162402X.2023.2207868

Figure Lengend Snippet: Combinatorial treatment with ReTARG fusion proteins potentiates cancer cell elimination. (a) Schematic representation of EGFR-ReTARG IE-1 fusion protein. The anti-EGFR Fab domain of EGFR-ReTARG IE-1 was constructed using published VH and VL gene sequences of mAb 425 . (b) Binding of EGFR-ReTARG IE-1 (0.5 μg/ml) toward EGFR pos DLD-1 cancer cells. Of note, DLD-1 cells are β2 M-deficient. (c) Cytotoxic capacity of anti‐CMV IE-1 CD8 pos T cells toward EGFR pos OvCAR-3 cancer cells at increasing E:T cell ratios in the presence (or absence) of EGFR-ReTARG IE-1 (100 ng/ml). Apoptotic cancer cell death was evaluated by Annexin-V/PI staining after 24 h. Cytotoxic capacity of a 1:1 mix of HLA‐B × 07:02‐restricted anti‐CMV pp65 CD8 pos T cells and HLA‐C × 07:02-restricted anti-CMV IE-1 CD8 pos T cells toward EpCAM pos /EGFR pos (d) OvCAR-3 and (e) A-431 cancer cells (E:T cell ratio = 1:1) in the presence (or absence) of EpCAM-ReTARG pp65 or/and EGFR-ReTARG IE-1 (both 100 ng/ml) using a conditionally fluorescent caspase-3/8 substrate. Quantification of green fluorescent cells over time is shown. (f) Cytotoxic capacity of freshly isolated PBMCs toward EpCAM pos /EGFR pos PC-3 M cancer cells (E:T cell ratio = 10:1–20:1) in the presence (or absence) of EpCAM-ReTARG pp65 or/and EGFR-ReTARG IE-1 (both 100 ng/ml). Apoptotic cancer cell death was evaluated by Annexin-V/PI staining after 4 d. (g) Capacity of EpCAM-ReTARG pp65 and EGFR-ReTARG IE-1 (both 100 ng/ml) to activate a 1:1 mix of anti‐CMV pp65 /anti-CMV IE-1 CD8 pos T cells in the presence of OvCAR-3 cancer cells (E:T cell ratio = 1:1). Conditioned culture media was collected after 24 h and T cell-secreted IFNγ was quantified by ELISA. (h) Capacity of EpCAM-ReTARG pp65 and EGFR-ReTARG IE-1 (both 100 ng/ml) to activate PBMCs in the presence of PC-3 M cancer cells (E:T cell ratio = 10:1). Conditioned culture media was collected after 4 d and T cell-secreted IFNγ was quantified by ELISA. The ex vivo-expanded T cells used were from CMV‐seropositive/HLA‐B × 07:02 pos donor #2. Graphs B, D, E, G and H show representative images with two technical replicates (mean ± SD). Graphs C and F: n = 3 (two technical replicates), mean ± SD are shown. Statistical analysis in C was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test. Statistical analysis in D-F was performed using two-way ANOVA followed by Bonferroni-Dunn post-hoc test (EpCAM-ReTARG pp65 + EGFR-ReTARG IE-1 vs EpCAM-ReTARG pp65 and EGFR-ReTARG IE-1 , respectively). Representatively, only the comparison with lower significance level is shown in the graph. Statistical analysis in G+H was performed using one-way ANOVA followed by Bonferroni-Dunn post-hoc test. (*p < .05, **p < .01, ***p < .001).

Article Snippet: PBMCs were stimulated with either recombinant CMV pp65 (Miltenyi Biotech) or T cell-activated IE-1 (Lophius Biosciences) protein solution for 2 d. Next, stimulated PBMCs were harvested, resuspended in fresh X-VIVO15 medium supplemented with 500 U/ml IL-2 (Immunotools), and cultured for an additional 4 d. Subsequently, CMV pp65 - and CMV IE-1 -stimulated PBMCs were cultured on a feeder layer of OvCAR-3.pp65 cells and OvCAR-3.IE-1 at an effector-to-target (E:T) cell ratio of 7.5:1.

Techniques: Construct, Binding Assay, Staining, Isolation, Enzyme-linked Immunosorbent Assay, Ex Vivo, Comparison

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells target citrullinated antigens in rheumatoid arthritis

doi: 10.1038/s41467-022-35264-8

Figure Lengend Snippet: a , b PBMCs (5 × 10 5 ) of ACPA+ RA patients were incubated with anti-CD3/28 antibodies ( n = 14), NP (influenza A)/ pp65 (CMV) protein (50 μM of each, n = 14), native vimentin protein (100 μM, n = 5), or citrullinated vimentin (cit-vimentin) protein (100 μM, n = 14) with or without anti-CD8 and/or HLA class I-blocking antibody (anti-CD8 Ab n = 6, anti-HLA class I Ab n = 6 or anti-CD8/ HLA class I Abs n = 10), or no stimulation ( n = 15) for 3 days. The percentage of Ki-67-expressing CD8 + ( a ) or CD4 + ( b ) T cells in ACPA+ RA PBMCs was measured by flow cytometry. For a , *** P < 0.0001, ** P = 0.026, ** P = 0.0021, # P = 0.0471, # P = 0.026, ## P = 0.0056 or ## P = 0.0021 . For b , *** P < 0.0001, or * P = 0.0383. c , d Native vimentin or cit–vimentin pulsed monocyte-derived dendritic cells (MoDCs) were cocultured with Cell Proliferation Dye eF450-labeled CD3 + T cells isolated from ACPA+ RA PBMCs ( n = 9) with or without anti-CD8/HLA class I-blocking antibody for 10 days, followed by flow cytometry analysis. Representative flow cytometry results ( c , left ) and quantification of the percentage of dye low proliferating CD8 + T cells ( c , right ) or CD4 + T cells ( d ). For c , *** P < 0.0001, ** P = 0.0054, ** P = 0.0015, # P = 0.0494 or # P = 0.0251. For d , *** P < 0.0001, * P = 0.0171, or # P = 0.0484. e Proliferation capacity of ACPA + RA CD8 + T cells based on proliferation dye eF450-labeled CD8 + T cells co-cultured with cit-vimentin or native vimentin-loaded MoDCs in the absence or presence of anti-CD8/HLA class I-blocking antibody for 10 days ( n = 4). ** P = 0.0011, ** P = 0.0027, # P = 0.0486, or ### P = 0.0007. Representative histograms ( left ) and quantification of the proliferating CD8 + T cells ( right ). Bars represent means ± SEM. * P < 0.05, ** P < 0.01, or *** P < 0.001 versus no treatment by unpaired t -test with two-tailed test; and # P < 0.05, ## P < 0.01, or ### P < 0.001 versus cit-vimentin treatment by unpaired t -test with two-tailed test. Source data are provided as a Source Data file. HC healthy control, ACPA anti-citrullinated protein antibodies, RA rheumatoid arthritis, Cit-Vim citrullinated vimentin.

Article Snippet: Within 4 h of blood collection, 1 ml of whole blood was incubated with 2 μg/ml anti-CD28 (BD Biosciences, 555725) and CD49d (BioLegend, 304302) antibodies, 50 μM each of recombinant influenza A H1N1 nucleoprotein (NP, Sino Biological, 11675-V08B) and CMV pp65 (Miltenyi Biotec, 130-091-824) protein, 100 μM of cit-vimentin (Cayman Chemical, 21942), 100 μM native vimentin (Cayman Chemical, 11234), or 1 μg/ml anti-CD3 antibodies (BD Biosciences, 566685) were added, followed by incubation at 37 °C for 6 h. In the last 5 h, Golgi inhibitor Brefeldin A (10 μg/ml) was added, and then addition of 20 mM EDTA for 15 min followed by FACS lysing solution (BD Biosciences, 349202) to lyse RBCs.

Techniques: Incubation, Blocking Assay, Expressing, Flow Cytometry, Derivative Assay, Labeling, Isolation, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: Cytotoxic CD8 + T cells target citrullinated antigens in rheumatoid arthritis

doi: 10.1038/s41467-022-35264-8

Figure Lengend Snippet: a Stimulation of fresh blood from ACPA+ RA ( n = 8, red bars) or HCs ( n = 7, blue bars) with anti-CD3/28 Abs, NP/pp65, cit- or native vimentin for 6 h with addition of Golgi inhibitor for the last 5 h. Quantification of CD69 + and GzmB + IFNγ + CD8 + T cells, and IFNγ + CD4 + T cells. For CD69 + CD8 + , * P = 0.049, + P = 0.0397, ** P = 0.001, ## P = 0.009, or ### P = 0.0008. For GzmB + IFNγ + CD8 + , + P = 0.0175, ** P = 0.0071, ## P = 0.0071, or ## P = 0.0037. For IFNγ + CD4 + , ++ P = 0.0065, + P = 0.0489, * P = 0.0155, # P = 0.0351, or ## P = 0.0038. b PBMCs of ACPA+ RA patients ( n = 10) were incubated with anti-CD3/28 Abs, NP/pp65 (50 μM of each), individual citrullinated proteins (100 μM), all citrullinated proteins (20 μM each), or all native proteins (20 μM each) for 16 h. Percentage of IFNγ or Granzyme B (GzmB) expressing CD8 + T cells measured by intracellular staining. c Quantification of IFNγ + or GzmB + IFNγ + expressing CD8 + T cells in ACPA+ RA PBMCs treated with cit- or native vimentin in a dose-dependent manner (1, 10 or 100 μM) ( n = 7). d Quantitative analysis of IFNγ + or GzmB + IFNγ + expressing CD8 + T cells in ACPA+ RA PBMCs stimulated with cit-vimentin in the presence or absence of anti-CD8 and/or HLA class I-blocking antibody ( n = 10). Data are presented as means ± SEM. For a , two-tailed unpaired t- test: * P < 0.05 or ** P < 0.01 versus no treatment in ACPA+ RA; + P < 0.05, or ++ P < 0 . 01 versus no treatment in HC; and # P < 0.05, ## P < 0.01, or ### P < 0.001 versus cit-vimentin in ACPA + RA. For b , two-tailed unpaired t -test: * P < 0.05, ** P < 0.01, or *** P < 0.001 versus no treatment; # P < 0.05, or ## P < 0.01 versus all native proteins. For c , two-tailed unpaired t -test: * P < 0.05, or ** P < 0.01 versus no treatment; # P < 0.05 for cit-vimentin 1 μM versus vimentin 1 μM; $ P < 0.05 for cit-vimen t in 10 μM versus native vimentin 10 μM by two-tailed unpaired t -test; + P < 0 . 05 for cit-vimentin 100 μM versus native vimentin 100 μM. For d , by two-tailed unpaired t -test: * P < 0.05, or ** P < 0.01 versus no treatment; # P < 0.05, or ## P < 0.01 versus cit-vimentin 100 μM. Source data are provided as a Source Data file. HC healthy control, ACPA anti-ci t rullinated protein antibodies, RA rheumatoid arthritis, Cit-Vim citrullinated vimentin.

Article Snippet: Within 4 h of blood collection, 1 ml of whole blood was incubated with 2 μg/ml anti-CD28 (BD Biosciences, 555725) and CD49d (BioLegend, 304302) antibodies, 50 μM each of recombinant influenza A H1N1 nucleoprotein (NP, Sino Biological, 11675-V08B) and CMV pp65 (Miltenyi Biotec, 130-091-824) protein, 100 μM of cit-vimentin (Cayman Chemical, 21942), 100 μM native vimentin (Cayman Chemical, 11234), or 1 μg/ml anti-CD3 antibodies (BD Biosciences, 566685) were added, followed by incubation at 37 °C for 6 h. In the last 5 h, Golgi inhibitor Brefeldin A (10 μg/ml) was added, and then addition of 20 mM EDTA for 15 min followed by FACS lysing solution (BD Biosciences, 349202) to lyse RBCs.

Techniques: Incubation, Expressing, Staining, Blocking Assay, Two Tailed Test