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A porcine model of acute rejection for cardiac transplantation. (A) Yucatan pigs are first genotyped for SLA haplotype. Donor-recipient pairs are selected where there is complete mismatch of all SLA class I and class II genes. The recipient pig begins receiving immunosuppression therapy three days prior to transplantation. (B) The heart is procured from the donor pig and undergoes ex vivo normothermic machine perfusion for two-hours. The recipient pig then undergoes abdominal heterotopic heart transplantation. Following transplantation, the recipient continues to receive immunosuppression therapy for 14-days, at which time (C) close observation begins for monitoring acute rejection driven primarily by cellular processes involving CD4 + and <t>CD8</t> + cells. (D) Assessments through ultrasound, CMR, histology, blood analysis, and cfDNA quantitation were used to monitor for acute rejection. (E) Observation was continued until fulminant rejection was reached.

Cd8α, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A porcine model of acute rejection for cardiac transplantation"

Article Title: A porcine model of acute rejection for cardiac transplantation

Journal: Frontiers in Cardiovascular Medicine

doi: 10.3389/fcvm.2025.1549377

Figure Legend Snippet: A porcine model of acute rejection for cardiac transplantation. (A) Yucatan pigs are first genotyped for SLA haplotype. Donor-recipient pairs are selected where there is complete mismatch of all SLA class I and class II genes. The recipient pig begins receiving immunosuppression therapy three days prior to transplantation. (B) The heart is procured from the donor pig and undergoes ex vivo normothermic machine perfusion for two-hours. The recipient pig then undergoes abdominal heterotopic heart transplantation. Following transplantation, the recipient continues to receive immunosuppression therapy for 14-days, at which time (C) close observation begins for monitoring acute rejection driven primarily by cellular processes involving CD4 + and CD8 + cells. (D) Assessments through ultrasound, CMR, histology, blood analysis, and cfDNA quantitation were used to monitor for acute rejection. (E) Observation was continued until fulminant rejection was reached.

Techniques Used: Transplantation Assay, Ex Vivo, Quantitation Assay

Histologic and functional changes of acute rejection. (A) Palpation grade was assessed 3–4 times daily for each animal during the observation period (peri-operative immunosuppression period shaded in blue). (B) Troponin levels were measured 2–3 times a week and were significantly greater at the time of fulminant acute rejection relative to POD 19-23 and baseline (Wilcoxon matched-pairs signed rank test). (C) Echocardiography was used as an adjunct to palpation grading to assess for cessation of cardiac activity and qualitative trending of LV wall thickness. (D) Cross sections were examined following explantation of the animal's native heart (top) and of the allograft (bottom). By the time of fulminant acute rejection there was pronounced LV hypertrophy. (E) There was also notable scarring and inflammation of the allograft (bottom) at time of explantation noted on gross pathology. (F) On H&E there was significant lymphocytic infiltration and myocardial degeneration; images shown were taken at 10× magnification. (G) Immunofluorescent staining of the allograft tissue for CD3 and CD8 identified cytotoxic T cell infiltration of tissues; images shown were taken at 20× magnification.

Figure Legend Snippet: Histologic and functional changes of acute rejection. (A) Palpation grade was assessed 3–4 times daily for each animal during the observation period (peri-operative immunosuppression period shaded in blue). (B) Troponin levels were measured 2–3 times a week and were significantly greater at the time of fulminant acute rejection relative to POD 19-23 and baseline (Wilcoxon matched-pairs signed rank test). (C) Echocardiography was used as an adjunct to palpation grading to assess for cessation of cardiac activity and qualitative trending of LV wall thickness. (D) Cross sections were examined following explantation of the animal's native heart (top) and of the allograft (bottom). By the time of fulminant acute rejection there was pronounced LV hypertrophy. (E) There was also notable scarring and inflammation of the allograft (bottom) at time of explantation noted on gross pathology. (F) On H&E there was significant lymphocytic infiltration and myocardial degeneration; images shown were taken at 10× magnification. (G) Immunofluorescent staining of the allograft tissue for CD3 and CD8 identified cytotoxic T cell infiltration of tissues; images shown were taken at 20× magnification.

Techniques Used: Functional Assay, Activity Assay, Staining

Expansion of T cells in the recipient animals’ circulation over time . Blood samples were collected over the course of each animal's observation period (A) Peripheral blood mononuclear cells (PBMC) were isolated (B) and used for immunophenotyping through flow cytometry (C) Overall cell population changes were pooled from Animals A–E and G using uniform manifold approximation and projection (UMAP) analysis (D) Three timepoints were assessed: baseline, 3 weeks post-transplantation, and at the time of cessation of graft activity. Relative to baseline, at the endpoint there was significant expansion of CD4 + (6.6%) and CD8 + (27.8%) T cells. There was also significant expansion of CD25 + T cells (14.9%), CD56 + natural killer cells (14.2%), and CD21 + B cells (3.2%). The changes in specific cell subtypes from baseline to endpoint are also shown as plots with the blue area corresponding to the baseline and the grey area corresponding to the endpoint (E) .

Figure Legend Snippet: Expansion of T cells in the recipient animals’ circulation over time . Blood samples were collected over the course of each animal's observation period (A) Peripheral blood mononuclear cells (PBMC) were isolated (B) and used for immunophenotyping through flow cytometry (C) Overall cell population changes were pooled from Animals A–E and G using uniform manifold approximation and projection (UMAP) analysis (D) Three timepoints were assessed: baseline, 3 weeks post-transplantation, and at the time of cessation of graft activity. Relative to baseline, at the endpoint there was significant expansion of CD4 + (6.6%) and CD8 + (27.8%) T cells. There was also significant expansion of CD25 + T cells (14.9%), CD56 + natural killer cells (14.2%), and CD21 + B cells (3.2%). The changes in specific cell subtypes from baseline to endpoint are also shown as plots with the blue area corresponding to the baseline and the grey area corresponding to the endpoint (E) .

Techniques Used: Isolation, Flow Cytometry, Transplantation Assay, Activity Assay

Image Search Results

Inhibition of NAT10 enhances the efficacy of anti–PD-L1 therapy in vivo . A Schematic diagram of the tumor model and treatment schedule with anti-PD-L1 administration. B–D Representative images ( B ), tumor growth curves ( C ), and tumor weights ( D ) of mouse 4T1 tumors from shNC, shNAT10, anti-PD-L1, or the combination of shNAT10 and anti-PD-L1 ( n = 6). E-F Representative mIF images of mouse tumor tissues showing staining for T cells ( E ), Tregs ( F ), and MDSCs ( F ) (scale bar, 20 μm). G - H ELISA analysis of GzmB (G) and IFN-γ (H) levels in the serum of mice from different groups. I Representative IHC staining of PD-L1, CD8, and GzmB in breast cancer tissues ( n = 220), with statistical analysis of staining intensity (scale bar, 200 μm). J Correlation of PD-L1 and CD8, and PD-L1 and GzmB protein levels in breast cancer tissues ( n = 220) assessed by IHC. All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting the NAT10-HDAC4 positive feedback loop counteracts immunosuppression in breast cancer

doi: 10.1186/s13046-025-03638-7

Figure Lengend Snippet: Inhibition of NAT10 enhances the efficacy of anti–PD-L1 therapy in vivo . A Schematic diagram of the tumor model and treatment schedule with anti-PD-L1 administration. B–D Representative images ( B ), tumor growth curves ( C ), and tumor weights ( D ) of mouse 4T1 tumors from shNC, shNAT10, anti-PD-L1, or the combination of shNAT10 and anti-PD-L1 ( n = 6). E-F Representative mIF images of mouse tumor tissues showing staining for T cells ( E ), Tregs ( F ), and MDSCs ( F ) (scale bar, 20 μm). G - H ELISA analysis of GzmB (G) and IFN-γ (H) levels in the serum of mice from different groups. I Representative IHC staining of PD-L1, CD8, and GzmB in breast cancer tissues ( n = 220), with statistical analysis of staining intensity (scale bar, 200 μm). J Correlation of PD-L1 and CD8, and PD-L1 and GzmB protein levels in breast cancer tissues ( n = 220) assessed by IHC. All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: In the CD8+ T cell depletion assay, mice were administered anti-Mouse CD8α antibody (Clone 2.43) (MedChemExpress, #HY-P990790, China) intraperitoneally at a dose of 300 μg daily for three consecutive days.

Techniques: Inhibition, In Vivo, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Changes in T cell subsets in mice after immunization. (A) Flow cytometry analysis of changes in CD4 + T cells in the hTSHR289, hTSHR290 and hTSHR410 groups after immunization. (B) Flow cytometry analysis of changes in CD8 + T cells in the hTSHR289, hTSHR290 and hTSHR410 groups after immunization. *P<0.05 (One-way ANOVA with Tukey's post hoc test). hTSHR, human thyroid-stimulating hormone receptor; ns, not significant.

Journal: Molecular Medicine Reports

Article Title: Expression, immunogenicity and clinical significance analysis of thyroid-stimulating hormone receptor fusion proteins

doi: 10.3892/mmr.2025.13639

Figure Lengend Snippet: Changes in T cell subsets in mice after immunization. (A) Flow cytometry analysis of changes in CD4 + T cells in the hTSHR289, hTSHR290 and hTSHR410 groups after immunization. (B) Flow cytometry analysis of changes in CD8 + T cells in the hTSHR289, hTSHR290 and hTSHR410 groups after immunization. *P<0.05 (One-way ANOVA with Tukey's post hoc test). hTSHR, human thyroid-stimulating hormone receptor; ns, not significant.

Article Snippet: The analyte detectors were as follows: CD3 + antibody (cat. no. 565643; Becton, Dickinson and Company), CD4 + antibody (cat. no. F21004A02; Multi Sciences Biotech), CD8 + antibody (cat. no. F2100801; Multi Sciences Biotech), CD25 antibody (cat. no. E-AB-F1102C; Wuhan Elabscience Biotechnology Co., Ltd.) and CD122 antibody (cat. no. E-AB-F1029D; Wuhan Elabscience Biotechnology Co., Ltd.).

Techniques: Flow Cytometry

Changes of specific regulatory T cell subsets in mice after immunization. (A) Flow cytometry analysis of changes in CD4 + CD25 + T cells in the hTSHR289, hTSHR290 and hTSHR410 groups after immunization. (B) Flow cytometry analysis of changes in CD8 + CD122 + T cells in the hTSHR289, hTSHR290 and hTSHR410 groups after immunization. *P<0.05 (One-way ANOVA with Tukey's post hoc test. hTSHR, human thyroid-stimulating hormone receptor.

Journal: Molecular Medicine Reports

Article Title: Expression, immunogenicity and clinical significance analysis of thyroid-stimulating hormone receptor fusion proteins

doi: 10.3892/mmr.2025.13639

Figure Lengend Snippet: Changes of specific regulatory T cell subsets in mice after immunization. (A) Flow cytometry analysis of changes in CD4 + CD25 + T cells in the hTSHR289, hTSHR290 and hTSHR410 groups after immunization. (B) Flow cytometry analysis of changes in CD8 + CD122 + T cells in the hTSHR289, hTSHR290 and hTSHR410 groups after immunization. *P<0.05 (One-way ANOVA with Tukey's post hoc test. hTSHR, human thyroid-stimulating hormone receptor.

Techniques: Flow Cytometry

Specific expression of 4-1BB and CCR8 on tumor-infiltrating Tregs (A) Online analysis of the human colorectal cancer database (public dataset GEO: GSE146771 http://cancer-pku.cn:3838/CRC_Leukocyte/ ). (B) qPCR analysis of the expression of 4-1BB and CCR8 in different T cell subsets in the spleen, draining lymph nodes and tumor. 6-8w mice were subcutaneously injected with 5×10 5 CT26, MC38, or B16F10 tumor cells. After two weeks, mice were euthanized, and tumors, spleens, and draining lymph nodes were harvested for flow cytometry analysis. n = 5–7/group. (C) Flow cytometry analysis showing 4-1BB expression on CD8 + T cells and CCR8 expression on Tregs in the spleen, draining lymph nodes, and tumor in the CT26 model. (D and E) Expression of 4-1BB/CCR8 on CD8 + T cell and Treg cell in tumors, spleens, and draining lymph nodes in the CT26, MC38 and B16F10 tumor models, as determined by flow cytometry statistical analysis. (D) 4-1BB; (E) CCR8. (F) Flow cytometry diagram showing co-expression of 4-1BB and CCR8 on CD8 + T cells and Tregs within the CT26 tumor. (G) Statistical analysis of 4-1BB + CCR8 + Tregs and 4-1BB + CCR8 + CD8 cells in CT26 tumors. (H) qPCR analysis of LAG3, TGF-β and Ki67 in different Treg subpopulations. (I) Tregs from tumour-bearing Foxp3-EGFP mice were isolated and sorted into three subsets based on CCR8 and 4-1BB expression: CCR8 - 4-1BB − , CCR8 - 4-1BB + and CCR8 + 4-1BB + . Each Treg subset was co-cultured with CellTrace Violet (CTV)-labeled CD8 + T cells at a 1:1 ratio. After 3 days, CD8 + T cell proliferation was analyzed by flow cytometry based on CTV dilution. The right panel shows the quantification of proliferating CD8 + T cells. The data from three independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Sp: spleen; DLN: draining lymph nodes.

Journal: iScience

Article Title: Bispecific targeting of 4-1BB and CCR8 boosts antitumor immunity via Ti-Treg depletion and CD8 + activation

doi: 10.1016/j.isci.2025.112829

Figure Lengend Snippet: Specific expression of 4-1BB and CCR8 on tumor-infiltrating Tregs (A) Online analysis of the human colorectal cancer database (public dataset GEO: GSE146771 http://cancer-pku.cn:3838/CRC_Leukocyte/ ). (B) qPCR analysis of the expression of 4-1BB and CCR8 in different T cell subsets in the spleen, draining lymph nodes and tumor. 6-8w mice were subcutaneously injected with 5×10 5 CT26, MC38, or B16F10 tumor cells. After two weeks, mice were euthanized, and tumors, spleens, and draining lymph nodes were harvested for flow cytometry analysis. n = 5–7/group. (C) Flow cytometry analysis showing 4-1BB expression on CD8 + T cells and CCR8 expression on Tregs in the spleen, draining lymph nodes, and tumor in the CT26 model. (D and E) Expression of 4-1BB/CCR8 on CD8 + T cell and Treg cell in tumors, spleens, and draining lymph nodes in the CT26, MC38 and B16F10 tumor models, as determined by flow cytometry statistical analysis. (D) 4-1BB; (E) CCR8. (F) Flow cytometry diagram showing co-expression of 4-1BB and CCR8 on CD8 + T cells and Tregs within the CT26 tumor. (G) Statistical analysis of 4-1BB + CCR8 + Tregs and 4-1BB + CCR8 + CD8 cells in CT26 tumors. (H) qPCR analysis of LAG3, TGF-β and Ki67 in different Treg subpopulations. (I) Tregs from tumour-bearing Foxp3-EGFP mice were isolated and sorted into three subsets based on CCR8 and 4-1BB expression: CCR8 - 4-1BB − , CCR8 - 4-1BB + and CCR8 + 4-1BB + . Each Treg subset was co-cultured with CellTrace Violet (CTV)-labeled CD8 + T cells at a 1:1 ratio. After 3 days, CD8 + T cell proliferation was analyzed by flow cytometry based on CTV dilution. The right panel shows the quantification of proliferating CD8 + T cells. The data from three independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Sp: spleen; DLN: draining lymph nodes.

Article Snippet: Anti-mouse CD8α , Selleck , Cat #A2102.

Techniques: Expressing, Injection, Flow Cytometry, Isolation, Cell Culture, Labeling

Development and characterization of the bsAb FRP303 (A) Schematic representation of the composition of FRP303. (B) In vitro analysis of the ADCC effect of the Vehicle (hIgG1), α-4-1BB,α-CCR8,FRP303 on CHO-m4-1BB, CHO-mCCR8, and CHO-m4-1BB-mCCR8 cell lines by using Jurkat-FcγRIIIa-luciferase. (C) The ADCC effect of 1 nM FRP303 on CHOK1, CHO-m4-1BB, CHO-mCCR8, and CHO-m4-1BB-mCCR8 cell lines. (D) Representative flow diagram showing the percentages of FRP303-binding Tregs and CD8 + T cells in tumors and Flow cytometric analysis. (E) Assessment of the effects of FRP303-mediated ADCC on tumor-infiltrating CD8 + T cells and Tregs. (F and G) CD8 + T cells were enriched from the spleens of WT mice, followed by treatment with 2 μg/mL of FRP303 for 48 h with re-stimulation using PMA, Ionomycin, and BFA for 4 h prior to sample collection. Flow cytometric analysis of IFN-γ and TNF-α in CD8 + T cells (F) and qPCR were performed (G). The data from three independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Journal: iScience

Article Title: Bispecific targeting of 4-1BB and CCR8 boosts antitumor immunity via Ti-Treg depletion and CD8 + activation

doi: 10.1016/j.isci.2025.112829

Figure Lengend Snippet: Development and characterization of the bsAb FRP303 (A) Schematic representation of the composition of FRP303. (B) In vitro analysis of the ADCC effect of the Vehicle (hIgG1), α-4-1BB,α-CCR8,FRP303 on CHO-m4-1BB, CHO-mCCR8, and CHO-m4-1BB-mCCR8 cell lines by using Jurkat-FcγRIIIa-luciferase. (C) The ADCC effect of 1 nM FRP303 on CHOK1, CHO-m4-1BB, CHO-mCCR8, and CHO-m4-1BB-mCCR8 cell lines. (D) Representative flow diagram showing the percentages of FRP303-binding Tregs and CD8 + T cells in tumors and Flow cytometric analysis. (E) Assessment of the effects of FRP303-mediated ADCC on tumor-infiltrating CD8 + T cells and Tregs. (F and G) CD8 + T cells were enriched from the spleens of WT mice, followed by treatment with 2 μg/mL of FRP303 for 48 h with re-stimulation using PMA, Ionomycin, and BFA for 4 h prior to sample collection. Flow cytometric analysis of IFN-γ and TNF-α in CD8 + T cells (F) and qPCR were performed (G). The data from three independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-mouse CD8α , Selleck , Cat #A2102.

Techniques: In Vitro, Luciferase, Binding Assay

FRP303 specifically eliminates Ti-Tregs and promotes CD8 + T cell function. (A–I) In the CT26 treatment model, flow cytometry was used to analyze changes in T cell subsets and their functions within the tumor microenvironment. n = 7–8/group (A) Flow cytometry contour plots of Treg cells and flow cytometry analysis of Treg cells in the CT26 tumor model. (B) The number of Treg cells per gram of tumor tissue. (C) The relative ratio of CD8 + T cells to Treg cells. (D) Flow cytometry contour plots of CD8 + T cells and flow cytometry analysis of CD8 + T cells in the CT26 tumor model. (E) The number of CD8 + T cells per gram of tumor tissue. (F) The proportion of effector CD8 + T cells (CD44 + CD62L − ). (G) Quantitative analysis of antitumor cytokine production (IFN-γ and TNF-α) by CD8 + T cells. (H) Quantitative analysis of antitumor cytokine production (IFN-γ and TNF-α) by Tconv cells. The data from at least two independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Journal: iScience

Article Title: Bispecific targeting of 4-1BB and CCR8 boosts antitumor immunity via Ti-Treg depletion and CD8 + activation

doi: 10.1016/j.isci.2025.112829

Figure Lengend Snippet: FRP303 specifically eliminates Ti-Tregs and promotes CD8 + T cell function. (A–I) In the CT26 treatment model, flow cytometry was used to analyze changes in T cell subsets and their functions within the tumor microenvironment. n = 7–8/group (A) Flow cytometry contour plots of Treg cells and flow cytometry analysis of Treg cells in the CT26 tumor model. (B) The number of Treg cells per gram of tumor tissue. (C) The relative ratio of CD8 + T cells to Treg cells. (D) Flow cytometry contour plots of CD8 + T cells and flow cytometry analysis of CD8 + T cells in the CT26 tumor model. (E) The number of CD8 + T cells per gram of tumor tissue. (F) The proportion of effector CD8 + T cells (CD44 + CD62L − ). (G) Quantitative analysis of antitumor cytokine production (IFN-γ and TNF-α) by CD8 + T cells. (H) Quantitative analysis of antitumor cytokine production (IFN-γ and TNF-α) by Tconv cells. The data from at least two independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-mouse CD8α , Selleck , Cat #A2102.

Techniques: Cell Function Assay, Flow Cytometry

FRP303 does not impair peripheral immune homeostasis and minimizes hepatotoxicity (A) Proportion of Treg cells (right) and CCR8 + Treg cells (left) in the thymus. n = 6/group. (B) Percentage of Treg (left) and CD8 + T cells (right) among CD3 + cells in peripheral blood and spleen of mice following FRP303 treatment. n = 7/group. (C) H&E staining analysis of the heart, liver, lungs, and kidneys in CT26 tumor-bearing mice following FRP303 treatment. (D) Immunohistochemical (IHC) staining for CD8 in liver tissues. (E) Serum ALT levels. The data from at least two independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Journal: iScience

Article Title: Bispecific targeting of 4-1BB and CCR8 boosts antitumor immunity via Ti-Treg depletion and CD8 + activation

doi: 10.1016/j.isci.2025.112829

Figure Lengend Snippet: FRP303 does not impair peripheral immune homeostasis and minimizes hepatotoxicity (A) Proportion of Treg cells (right) and CCR8 + Treg cells (left) in the thymus. n = 6/group. (B) Percentage of Treg (left) and CD8 + T cells (right) among CD3 + cells in peripheral blood and spleen of mice following FRP303 treatment. n = 7/group. (C) H&E staining analysis of the heart, liver, lungs, and kidneys in CT26 tumor-bearing mice following FRP303 treatment. (D) Immunohistochemical (IHC) staining for CD8 in liver tissues. (E) Serum ALT levels. The data from at least two independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-mouse CD8α , Selleck , Cat #A2102.

Techniques: Staining, Immunohistochemical staining, Immunohistochemistry

FRP303 is dependent on CD8 + T cells to exert antitumor effects and generate immune memory (A) Experimental scheme of FRP303 treatment in MC38 tumor–bearing Rag1 −/− mice; n = 6/group. (B) MC38 tumor growth curve. (C) MC38 tumor weight. (D) Experimental scheme of CD8 depletion in CT26 tumor–bearing mice, n = 6/group. (E) CT26 tumor growth curve. (F) CT26 tumor weight. (G) Schematic diagram of the antitumor immune memory in the CT26 tumor model with FRP303 treatment. After 10 mg/kg FRP303 treatment on Days 7, 10, 13, and 16 post tumor inoculation and when the tumors were eradicated, the mice were re-challenged with 5×10 5 CT26 cells without further treatment to assess the immune memory response on Day 54. (H) Tumor growth curve of the antitumor immune memory in the CT26 tumor model. On Day 72, the mice were euthanized, and peripheral blood and draining lymph nodes were collected for flow cytometry analysis of immune memory-related phenotypes. (I) The proportion of CD127 + CD8 + T cells. (J) Flow cytometry statistical analysis of the proportions of Tn (CD44 − CD62L + ), Tem (CD44 + CD62L − ) and Tcm (CD44 + CD62L + ) cells in peripheral blood. (K) Flow cytometry statistical analysis of the proportions of Tn (CD44 − CD62L + ), Tem (CD44 + CD62L − ), and Tcm (CD44 + CD62L + ) cells in draining lymph nodes (DLN). The data from two independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Journal: iScience

Article Title: Bispecific targeting of 4-1BB and CCR8 boosts antitumor immunity via Ti-Treg depletion and CD8 + activation

doi: 10.1016/j.isci.2025.112829

Figure Lengend Snippet: FRP303 is dependent on CD8 + T cells to exert antitumor effects and generate immune memory (A) Experimental scheme of FRP303 treatment in MC38 tumor–bearing Rag1 −/− mice; n = 6/group. (B) MC38 tumor growth curve. (C) MC38 tumor weight. (D) Experimental scheme of CD8 depletion in CT26 tumor–bearing mice, n = 6/group. (E) CT26 tumor growth curve. (F) CT26 tumor weight. (G) Schematic diagram of the antitumor immune memory in the CT26 tumor model with FRP303 treatment. After 10 mg/kg FRP303 treatment on Days 7, 10, 13, and 16 post tumor inoculation and when the tumors were eradicated, the mice were re-challenged with 5×10 5 CT26 cells without further treatment to assess the immune memory response on Day 54. (H) Tumor growth curve of the antitumor immune memory in the CT26 tumor model. On Day 72, the mice were euthanized, and peripheral blood and draining lymph nodes were collected for flow cytometry analysis of immune memory-related phenotypes. (I) The proportion of CD127 + CD8 + T cells. (J) Flow cytometry statistical analysis of the proportions of Tn (CD44 − CD62L + ), Tem (CD44 + CD62L − ) and Tcm (CD44 + CD62L + ) cells in peripheral blood. (K) Flow cytometry statistical analysis of the proportions of Tn (CD44 − CD62L + ), Tem (CD44 + CD62L − ), and Tcm (CD44 + CD62L + ) cells in draining lymph nodes (DLN). The data from two independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-mouse CD8α , Selleck , Cat #A2102.

Techniques: Flow Cytometry

FRP303 exhibits significant synergistic antitumor effects with PD-1 antibodies (A) Schematic diagram of the effects of treatment with FRP303 and an anti-PD-1 antibody on a murine CT26 tumor model; n = 6/group. (B) CT26 tumor growth curve. (C) CT26 tumor weight. (D) Schematic diagram of the effects of treatment with FRP303 and an anti-PD-1 antibody on a murine B16F10 tumor model, n = 6–7/group. (E) B16F10 tumor growth curve. (F) B16F10 tumor weight. (G–K) Flow cytometric analysis of changes in T cell subsets and effector T cell function within the tumor microenvironment following combination treatment of FRP303 and PD-1 antibody in B16F10 melanoma. (G) Percentage of Treg (left) and CD8 + T cells (right) among CD3 + T cells. (H) The ratio of CD8 + T cells/Tregs. (I) Statistical analysis of CD44 + CD62L − effector CD8 + T cells. (J) Quantitative analysis of antitumor cytokine production (IFN-γ and TNF-α) by CD8 + T cells. (K) Quantitative analysis of antitumor cytokine production (IFN-γ and TNF-α) by Tconv cells. The data from two independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Journal: iScience

Article Title: Bispecific targeting of 4-1BB and CCR8 boosts antitumor immunity via Ti-Treg depletion and CD8 + activation

doi: 10.1016/j.isci.2025.112829

Figure Lengend Snippet: FRP303 exhibits significant synergistic antitumor effects with PD-1 antibodies (A) Schematic diagram of the effects of treatment with FRP303 and an anti-PD-1 antibody on a murine CT26 tumor model; n = 6/group. (B) CT26 tumor growth curve. (C) CT26 tumor weight. (D) Schematic diagram of the effects of treatment with FRP303 and an anti-PD-1 antibody on a murine B16F10 tumor model, n = 6–7/group. (E) B16F10 tumor growth curve. (F) B16F10 tumor weight. (G–K) Flow cytometric analysis of changes in T cell subsets and effector T cell function within the tumor microenvironment following combination treatment of FRP303 and PD-1 antibody in B16F10 melanoma. (G) Percentage of Treg (left) and CD8 + T cells (right) among CD3 + T cells. (H) The ratio of CD8 + T cells/Tregs. (I) Statistical analysis of CD44 + CD62L − effector CD8 + T cells. (J) Quantitative analysis of antitumor cytokine production (IFN-γ and TNF-α) by CD8 + T cells. (K) Quantitative analysis of antitumor cytokine production (IFN-γ and TNF-α) by Tconv cells. The data from two independent experiments were calculated and are presented as the means ± SEMs. ns, p > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-mouse CD8α , Selleck , Cat #A2102.

Techniques: Cell Function Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: A porcine model of acute rejection for cardiac transplantation

doi: 10.3389/fcvm.2025.1549377

Figure Lengend Snippet: A porcine model of acute rejection for cardiac transplantation. (A) Yucatan pigs are first genotyped for SLA haplotype. Donor-recipient pairs are selected where there is complete mismatch of all SLA class I and class II genes. The recipient pig begins receiving immunosuppression therapy three days prior to transplantation. (B) The heart is procured from the donor pig and undergoes ex vivo normothermic machine perfusion for two-hours. The recipient pig then undergoes abdominal heterotopic heart transplantation. Following transplantation, the recipient continues to receive immunosuppression therapy for 14-days, at which time (C) close observation begins for monitoring acute rejection driven primarily by cellular processes involving CD4 + and CD8 + cells. (D) Assessments through ultrasound, CMR, histology, blood analysis, and cfDNA quantitation were used to monitor for acute rejection. (E) Observation was continued until fulminant rejection was reached.

Article Snippet: OCT embedded frozen 10 μm thick sections were stained for both CD3ɛ (Invitrogen MA1-90582, mouse, 1:150) to identify the presence of T cells, and CD8α (BioRad MCA6048GA, rabbit, 1:150) to identify the subset of cytotoxic T cells.

Techniques: Transplantation Assay, Ex Vivo, Quantitation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: A porcine model of acute rejection for cardiac transplantation

doi: 10.3389/fcvm.2025.1549377

Figure Lengend Snippet: Histologic and functional changes of acute rejection. (A) Palpation grade was assessed 3–4 times daily for each animal during the observation period (peri-operative immunosuppression period shaded in blue). (B) Troponin levels were measured 2–3 times a week and were significantly greater at the time of fulminant acute rejection relative to POD 19-23 and baseline (Wilcoxon matched-pairs signed rank test). (C) Echocardiography was used as an adjunct to palpation grading to assess for cessation of cardiac activity and qualitative trending of LV wall thickness. (D) Cross sections were examined following explantation of the animal's native heart (top) and of the allograft (bottom). By the time of fulminant acute rejection there was pronounced LV hypertrophy. (E) There was also notable scarring and inflammation of the allograft (bottom) at time of explantation noted on gross pathology. (F) On H&E there was significant lymphocytic infiltration and myocardial degeneration; images shown were taken at 10× magnification. (G) Immunofluorescent staining of the allograft tissue for CD3 and CD8 identified cytotoxic T cell infiltration of tissues; images shown were taken at 20× magnification.

Techniques: Functional Assay, Activity Assay, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: A porcine model of acute rejection for cardiac transplantation

doi: 10.3389/fcvm.2025.1549377

Figure Lengend Snippet: Expansion of T cells in the recipient animals’ circulation over time . Blood samples were collected over the course of each animal's observation period (A) Peripheral blood mononuclear cells (PBMC) were isolated (B) and used for immunophenotyping through flow cytometry (C) Overall cell population changes were pooled from Animals A–E and G using uniform manifold approximation and projection (UMAP) analysis (D) Three timepoints were assessed: baseline, 3 weeks post-transplantation, and at the time of cessation of graft activity. Relative to baseline, at the endpoint there was significant expansion of CD4 + (6.6%) and CD8 + (27.8%) T cells. There was also significant expansion of CD25 + T cells (14.9%), CD56 + natural killer cells (14.2%), and CD21 + B cells (3.2%). The changes in specific cell subtypes from baseline to endpoint are also shown as plots with the blue area corresponding to the baseline and the grey area corresponding to the endpoint (E) .

Techniques: Isolation, Flow Cytometry, Transplantation Assay, Activity Assay

Activation of CD8 + T cells primed by adjuvanted OVA via activated KikGR-red cells (A) Experimental design: the hair-clipped skin of KikGR mice was exposed to 436 nm violet light at an intensity of 400 mW/cm 2 for 4 min. Three hours later, the photoconverted skin site was treated with CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS as a vehicle control. Forty-eight hours post-administration, KikGR-red cells, MHCII hi CD11c med Mig DCs, and MHCII med CD11c hi Res DCs were isolated from the dLNs using FACS sorting. Naive OT-I CD8 + T cells at 5 × 10 4 per well isolated from OT-I mice were used to co-culture with either 5 × 10 3 of KikGR-red, MHC-II hi CD11c med Mig DCs, or MHC-II med CD11c hi Res DCs per well, respectively. (B–D) CD8 + T cell activation: three days post-co-culture, the number of CFSE lo CD45.1 + CD3 + CD8 + TCRVα2 + T cells co-cultured with KikGR-red cells (B), MHC-II hi CD11c med Mig DCs (C), or MHC-II med CD11c hi Res DCs (D) was measured by FACS. Data shown in (B) and (D) are representative of two independent experiments with 3–4 mice in each experiment (Alum + OVA, n = 4; MF59 + OVA, n = 3; CR108 + OVA, n = 4) (mean ± SEM). Data shown in (C) are representative of two independent experiments with 6–8 mice in each experiment (Alum + OVA, n = 7; MF59 + OVA, n = 8; CR108 + OVA, n = 6) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (B, C, D).

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Activation of CD8 + T cells primed by adjuvanted OVA via activated KikGR-red cells (A) Experimental design: the hair-clipped skin of KikGR mice was exposed to 436 nm violet light at an intensity of 400 mW/cm 2 for 4 min. Three hours later, the photoconverted skin site was treated with CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS as a vehicle control. Forty-eight hours post-administration, KikGR-red cells, MHCII hi CD11c med Mig DCs, and MHCII med CD11c hi Res DCs were isolated from the dLNs using FACS sorting. Naive OT-I CD8 + T cells at 5 × 10 4 per well isolated from OT-I mice were used to co-culture with either 5 × 10 3 of KikGR-red, MHC-II hi CD11c med Mig DCs, or MHC-II med CD11c hi Res DCs per well, respectively. (B–D) CD8 + T cell activation: three days post-co-culture, the number of CFSE lo CD45.1 + CD3 + CD8 + TCRVα2 + T cells co-cultured with KikGR-red cells (B), MHC-II hi CD11c med Mig DCs (C), or MHC-II med CD11c hi Res DCs (D) was measured by FACS. Data shown in (B) and (D) are representative of two independent experiments with 3–4 mice in each experiment (Alum + OVA, n = 4; MF59 + OVA, n = 3; CR108 + OVA, n = 4) (mean ± SEM). Data shown in (C) are representative of two independent experiments with 6–8 mice in each experiment (Alum + OVA, n = 7; MF59 + OVA, n = 8; CR108 + OVA, n = 6) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (B, C, D).

Article Snippet: InVivoMAb Rat anti-mouse CD8α IgG2b; clone L3 , BioXcell , Cat# BE0061; RRID: AB_1125541.

Techniques: Activation Assay, Control, Isolation, Co-Culture Assay, Cell Culture

Activation of CD8 + T Cells following immunization with various adjuvanted OVA (A) Experimental design: CFSE-labeled OT-I CD3 + CD8 + T cells (1 × 10 6 ) were isolated from donor animals and adoptively transferred intravenously into naive CD45.2 C57BL/six mice on day 0. The recipient mice were then immunized subcutaneously (s.c.) on day 1 with one of the following: CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS as a vehicle control. Three days post-immunization, CD45.1 + TCRVα2 + CD8 + T cell proliferation was assessed within draining lymph nodes (dLNs), non-draining lymph nodes (non-dLNs), and spleen via CFSE-dilution using flow cytometry. (B, C, and D) CFSE-dilution analysis in dLNs: histogram overlays depict CFSE-dilution of CD45.1 + TCRVα2 + CD8 + T cells in dLNs for each immunization group, illustrating the extent of T cell proliferation (B). (C) Proliferation index calculation: The T cell proliferation index was determined to quantify the overall proliferation response across different immunizations. (D) Proliferation in CFSE-division peaks: the percentage of CD45.1 + TCRVα2 + CD8 + T cells in each CFSE-division peak within dLNs was analyzed. Statistical significances: # p < 0.05, and #### p < 0.0001 (CR108 + OVA versus Alum + OVA), ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (CR108 + OVA versus MF59 + OVA) by Student’s t test, respectively. (E) Proliferative profiles in lymphoid organs: histogram overlays showing CFSE-dilution profiles of CD45.1 + TCRVα2 + CD8 + T cells in dLNs, non-dLNs, and spleens for all experimental groups, highlighting the differential T cell proliferation across these tissues. (F, G, and H) CD8 T cell proliferation in lymphoid tissues: the percentages of CD45.1 + TCRVα2 + CD8 + T cells in dLNs (F), non-dLNs (G), and spleens (H) were quantified. Data shown are representative of two independent experiments with 3–4 mice in each experiment (PBS, n = 3; OVA, n = 4; Alum + OVA, n = 4; MF59 + OVA, n = 4; CR108 + OVA, n = 4) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (C, F, G, H).

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Activation of CD8 + T Cells following immunization with various adjuvanted OVA (A) Experimental design: CFSE-labeled OT-I CD3 + CD8 + T cells (1 × 10 6 ) were isolated from donor animals and adoptively transferred intravenously into naive CD45.2 C57BL/six mice on day 0. The recipient mice were then immunized subcutaneously (s.c.) on day 1 with one of the following: CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS as a vehicle control. Three days post-immunization, CD45.1 + TCRVα2 + CD8 + T cell proliferation was assessed within draining lymph nodes (dLNs), non-draining lymph nodes (non-dLNs), and spleen via CFSE-dilution using flow cytometry. (B, C, and D) CFSE-dilution analysis in dLNs: histogram overlays depict CFSE-dilution of CD45.1 + TCRVα2 + CD8 + T cells in dLNs for each immunization group, illustrating the extent of T cell proliferation (B). (C) Proliferation index calculation: The T cell proliferation index was determined to quantify the overall proliferation response across different immunizations. (D) Proliferation in CFSE-division peaks: the percentage of CD45.1 + TCRVα2 + CD8 + T cells in each CFSE-division peak within dLNs was analyzed. Statistical significances: # p < 0.05, and #### p < 0.0001 (CR108 + OVA versus Alum + OVA), ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (CR108 + OVA versus MF59 + OVA) by Student’s t test, respectively. (E) Proliferative profiles in lymphoid organs: histogram overlays showing CFSE-dilution profiles of CD45.1 + TCRVα2 + CD8 + T cells in dLNs, non-dLNs, and spleens for all experimental groups, highlighting the differential T cell proliferation across these tissues. (F, G, and H) CD8 T cell proliferation in lymphoid tissues: the percentages of CD45.1 + TCRVα2 + CD8 + T cells in dLNs (F), non-dLNs (G), and spleens (H) were quantified. Data shown are representative of two independent experiments with 3–4 mice in each experiment (PBS, n = 3; OVA, n = 4; Alum + OVA, n = 4; MF59 + OVA, n = 4; CR108 + OVA, n = 4) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (C, F, G, H).

Article Snippet: InVivoMAb Rat anti-mouse CD8α IgG2b; clone L3 , BioXcell , Cat# BE0061; RRID: AB_1125541.

Techniques: Activation Assay, Labeling, Isolation, Control, Flow Cytometry

Induction of anti-tumor immune responses by various adjuvanted OVA (A–E) Schematic of representation of the experimental design. Naive female C57BL/six mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached to approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 4, and 7 (A). Average growth curves shown are mean ± SEM from two independent experiments (PBS, n = 8; OVA, n = 8; Alum + OVA, n = 8; MF59 + OVA, n = 8; CR108 + OVA, n = 9) (B). Statistical significances: # (CR108 + OVA vs. Alum + OVA, p < 0.05), ##(CR108 + OVA vs. Alum + OVA, p < 0.01) ∗(CR108 + OVA vs. MF59 + OVA, p < 0.05), and ns (CR108 + OVA vs. MF59 + OVA, not significant) by Student’s t test (C). The tumor weights were determined for each group. Quantification of tumor-infiltrating CD45 + CD3 + CD8 + T cells (D), and CD45 + CD3 + CD4 + T cells were determined by FACS at the end of the experiment (E) ( A). Data shown are representative of two independent experiments with four mice in each experiment (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (C, D, E). (F) In vivo CTL response measurement: C57BL/six mice were subcutaneously immunized with CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS on days 0 and 14. On day 21 post-first immunization. Cells pulsed with OVA 257–264 peptides were stained with CFSE high , while unpulsed cells were stained with CFSE low . CFSE high , and CFSE low cells were mixed in 1:1 and then intravenously transferred into the immunized mice. Twenty hours later, splenocytes were analyzed by flow cytometry to assess OVA-specific lysis. Data shown are representative of two independent experiments with 5–6 mice in each experiment (PBS, n = 6; OVA, n = 6; Alum + OVA, n = 6; MF59 + OVA, n = 6; CR108 + OVA, n = 5) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by One-way ANOVA. (G–I) Schematic representation of the treatment experimental design. Naive female C57BL/6 mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 7, and 14 (G). Average tumor growth curves (H) and the survival rates were measured until day 17 (I). Data shown are mean ± SEM from two independent experiments (untreated, n = 9; OVA, n = 10; Alum + OVA, n = 9; MF59 + OVA, n = 10; CR108 + OVA, n = 9). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (H). (J–L)Experimental design for α-CD4 or α-CD8 blocking. naive female C57BL/six mice were inoculated s.c. with 5 × 10 5 E.G7-OVA cells in the right flank. Once tumors reached 20 mm 3 , mice were intraperitoneally (i.p.) treated with 100 μg α-CD4 (GK1.5, BioXcell), 100 μg α-CD8 (2.43, BioXcell), or 100 μg rat IgG2b isotype control (LTF-2, BioXcell) on days 0, 5, 10, and 15. CR108 + OVA or PBS (untreated control) was administered near the tumor site on days 1, 7, and 13 (J). Average tumor growth curves (K) and survival rates (L) were determined. Data shown are mean ± SEM from two independent experiments (α-CD8 CR108 + OVA, n = 7; α-CD4 CR108 + OVA, n = 8; isotype CR108 + OVA, n = 7). Statistical significance: ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (K).

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Induction of anti-tumor immune responses by various adjuvanted OVA (A–E) Schematic of representation of the experimental design. Naive female C57BL/six mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached to approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 4, and 7 (A). Average growth curves shown are mean ± SEM from two independent experiments (PBS, n = 8; OVA, n = 8; Alum + OVA, n = 8; MF59 + OVA, n = 8; CR108 + OVA, n = 9) (B). Statistical significances: # (CR108 + OVA vs. Alum + OVA, p < 0.05), ##(CR108 + OVA vs. Alum + OVA, p < 0.01) ∗(CR108 + OVA vs. MF59 + OVA, p < 0.05), and ns (CR108 + OVA vs. MF59 + OVA, not significant) by Student’s t test (C). The tumor weights were determined for each group. Quantification of tumor-infiltrating CD45 + CD3 + CD8 + T cells (D), and CD45 + CD3 + CD4 + T cells were determined by FACS at the end of the experiment (E) ( A). Data shown are representative of two independent experiments with four mice in each experiment (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (C, D, E). (F) In vivo CTL response measurement: C57BL/six mice were subcutaneously immunized with CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS on days 0 and 14. On day 21 post-first immunization. Cells pulsed with OVA 257–264 peptides were stained with CFSE high , while unpulsed cells were stained with CFSE low . CFSE high , and CFSE low cells were mixed in 1:1 and then intravenously transferred into the immunized mice. Twenty hours later, splenocytes were analyzed by flow cytometry to assess OVA-specific lysis. Data shown are representative of two independent experiments with 5–6 mice in each experiment (PBS, n = 6; OVA, n = 6; Alum + OVA, n = 6; MF59 + OVA, n = 6; CR108 + OVA, n = 5) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by One-way ANOVA. (G–I) Schematic representation of the treatment experimental design. Naive female C57BL/6 mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 7, and 14 (G). Average tumor growth curves (H) and the survival rates were measured until day 17 (I). Data shown are mean ± SEM from two independent experiments (untreated, n = 9; OVA, n = 10; Alum + OVA, n = 9; MF59 + OVA, n = 10; CR108 + OVA, n = 9). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (H). (J–L)Experimental design for α-CD4 or α-CD8 blocking. naive female C57BL/six mice were inoculated s.c. with 5 × 10 5 E.G7-OVA cells in the right flank. Once tumors reached 20 mm 3 , mice were intraperitoneally (i.p.) treated with 100 μg α-CD4 (GK1.5, BioXcell), 100 μg α-CD8 (2.43, BioXcell), or 100 μg rat IgG2b isotype control (LTF-2, BioXcell) on days 0, 5, 10, and 15. CR108 + OVA or PBS (untreated control) was administered near the tumor site on days 1, 7, and 13 (J). Average tumor growth curves (K) and survival rates (L) were determined. Data shown are mean ± SEM from two independent experiments (α-CD8 CR108 + OVA, n = 7; α-CD4 CR108 + OVA, n = 8; isotype CR108 + OVA, n = 7). Statistical significance: ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (K).

Article Snippet: InVivoMAb Rat anti-mouse CD8α IgG2b; clone L3 , BioXcell , Cat# BE0061; RRID: AB_1125541.

Techniques: Control, In Vivo, Staining, Flow Cytometry, Lysis, Blocking Assay

Loss of CD8 + T cell activation due to CCR7 blockage affecting Mig DCs recruitment (A–C) Experimental procedure: the hair-clipped skin of KikGR mice was exposed to 436 nm violet light at an intensity of 400 mW/cm 2 for 4 min. Three hours later, 10 μg of α-CCR7 (4B12, R&D) blocking antibody per mouse was injected subcutaneously at the photoconverted skin site, while 10 μg of rat IgG2a antibody (2A3, BioXcell) per mouse was used as an isotype control. Six hours later, CR108 or MF59 plus OVA were administered subcutaneously at the α-CCR7 injection site. Forty-eight hours after administration, the numbers of (A) MHCII hi CD11c med Mig DCs, (B) KikGR-red cells, and (C) MHCII med CD11c hi Res DCs in the dLNs were measured by FACS. Data shown are representative of two independent experiments with three mice in each experiment (mean ± SEM). ∗ p < 0.05, ns (not significant) by Student’s t test (A, B, C). (D–H) Schematic of Experimental Design: CFSE-labeled OTI CD8 + T cells (1 × 10 6 ) were transferred into naive C57BL/six mice. One day later, the mice were subcutaneously injected with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control. Subsequently, the mice were immunized at the antibody-injected site with CR108 or MF59 plus OVA, and PBS as a vehicle control. Three days post-immunization, CFSE lo CD45.1 + CD3 + CD8 + TCRVα2 + T cells in dLNs, non-dLNs, and spleens were analyzed by FACS (D). Histogram analysis: histogram overlays of CFSE-dilution in CD45.1 + TCRVα2 + CD8 + T cells from dLNs, non-dLNs, and spleens were presented. (E) Quantitative Analysis: The number of CFSE lo CD45.1 + TCRVα2 + CD8 + T cells in dLNs (F), non-dLNs (G), and spleens (H) was quantified. Data shown are representative of two independent experiments with 3–4 mice in each experiment (mean ± SEM) (isotype CR108 + OVA, n = 4; α-CCR7 CR108 + OVA, n = 4; isotype MF59 + OVA, n = 3; α-CCR7 MF59 + OVA, n = 4; PBS, n = 4). ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns, not significant by Student’s t test (F, G, H). (I) Experimental design for α-CCR7 blocking: naive female C57BL/six mice were inoculated subcutaneously with E.G7-OVA cells (1 × 10 6 ) in the right flank, and tumor growth was monitored until the volume reached 20 mm 3 . To block Mig DCs migration, mice bearing E.G7-OVA tumors were treated subcutaneously with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control on days 1, 7, and 13. CR108 plus OVA were administered near the tumor sites, and PBS as a vehicle control (untreated), on days 1, 7, and 13. Tumor Growth Curves: Average tumor growth curves (α-CCR7 CR108 + OVA, n = 5; isotype CR108 + OVA, n = 4) were determined. Data shown are mean ± SEM from two independent experiments. ∗ p < 0.05 by two-way ANOVA.

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Loss of CD8 + T cell activation due to CCR7 blockage affecting Mig DCs recruitment (A–C) Experimental procedure: the hair-clipped skin of KikGR mice was exposed to 436 nm violet light at an intensity of 400 mW/cm 2 for 4 min. Three hours later, 10 μg of α-CCR7 (4B12, R&D) blocking antibody per mouse was injected subcutaneously at the photoconverted skin site, while 10 μg of rat IgG2a antibody (2A3, BioXcell) per mouse was used as an isotype control. Six hours later, CR108 or MF59 plus OVA were administered subcutaneously at the α-CCR7 injection site. Forty-eight hours after administration, the numbers of (A) MHCII hi CD11c med Mig DCs, (B) KikGR-red cells, and (C) MHCII med CD11c hi Res DCs in the dLNs were measured by FACS. Data shown are representative of two independent experiments with three mice in each experiment (mean ± SEM). ∗ p < 0.05, ns (not significant) by Student’s t test (A, B, C). (D–H) Schematic of Experimental Design: CFSE-labeled OTI CD8 + T cells (1 × 10 6 ) were transferred into naive C57BL/six mice. One day later, the mice were subcutaneously injected with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control. Subsequently, the mice were immunized at the antibody-injected site with CR108 or MF59 plus OVA, and PBS as a vehicle control. Three days post-immunization, CFSE lo CD45.1 + CD3 + CD8 + TCRVα2 + T cells in dLNs, non-dLNs, and spleens were analyzed by FACS (D). Histogram analysis: histogram overlays of CFSE-dilution in CD45.1 + TCRVα2 + CD8 + T cells from dLNs, non-dLNs, and spleens were presented. (E) Quantitative Analysis: The number of CFSE lo CD45.1 + TCRVα2 + CD8 + T cells in dLNs (F), non-dLNs (G), and spleens (H) was quantified. Data shown are representative of two independent experiments with 3–4 mice in each experiment (mean ± SEM) (isotype CR108 + OVA, n = 4; α-CCR7 CR108 + OVA, n = 4; isotype MF59 + OVA, n = 3; α-CCR7 MF59 + OVA, n = 4; PBS, n = 4). ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns, not significant by Student’s t test (F, G, H). (I) Experimental design for α-CCR7 blocking: naive female C57BL/six mice were inoculated subcutaneously with E.G7-OVA cells (1 × 10 6 ) in the right flank, and tumor growth was monitored until the volume reached 20 mm 3 . To block Mig DCs migration, mice bearing E.G7-OVA tumors were treated subcutaneously with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control on days 1, 7, and 13. CR108 plus OVA were administered near the tumor sites, and PBS as a vehicle control (untreated), on days 1, 7, and 13. Tumor Growth Curves: Average tumor growth curves (α-CCR7 CR108 + OVA, n = 5; isotype CR108 + OVA, n = 4) were determined. Data shown are mean ± SEM from two independent experiments. ∗ p < 0.05 by two-way ANOVA.

Article Snippet: InVivoMAb Rat anti-mouse CD8α IgG2b; clone L3 , BioXcell , Cat# BE0061; RRID: AB_1125541.

Techniques: Activation Assay, Blocking Assay, Injection, Control, Labeling, Migration

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