Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Memory CD8 + T cells with a resident phenotype are present in murine BM after acute systemic infection. (A–C) Spleen and BM were analyzed for virus‐specific CD8 T cells, 172 days after systemic infection with LCMV Armstrong. Data are shown for one representative experiment with n = 4 mice, out of three independent experiments; (A) Frequency of LCMV GP 33‐41 ‐specific and NP 396‐404 ‐specific CD8 + T cells of all CD44 + CD8 + T cells (average + SD); (B) Representative FACS staining for CD69 and CD62L on LCMV GP 33‐41 ‐specific CD44 + CD8 + T cells from spleen and BM; (C) Paired analysis for the percentage of CD62L − CD69 + cells of all LCMV GP 33‐41 ‐specific or NP 396‐404 ‐specific memory CD8 + T cells in spleen versus BM. (D–F) Analysis of OVA‐specific CD8 T cells in spleen and BM of WT mice, 49 days after systemic infection with Listeria‐OVA. Data are shown for one representative experiment with n = 9 mice, out of two independent experiments; (D) Frequency of OVA 257‐264 ‐specific CD8 + T cells of all CD44 + CD8 + T cells (average + SD); (E) Representative FACS staining for CD69 and CD62L on OVA 257‐264 ‐specific CD44 + CD8 + T cells from spleen and BM; (F) Paired analysis for the percentage of CD62L + CD69 + cells of all OVA 257‐264 ‐specific memory CD8 + T cells in spleen vs BM. (G–I) Expression levels of (G) CXCR3, (H) CXCR6, and (I) CX 3 CR1 in D b ‐GP 33‐41 ‐specific memory CD8 + T cell subsets in BM, 60 days after systemic infection with LCMV Armstrong. Data are shown for one representative experiment with n = 10 mice, out of two independent experiments. Data were analyzed by two‐tailed t ‐test, with matching (D, F), one‐way ANOVA followed by Tukey's multiple comparisons test (G–I) and two‐way ANOVA, with matching, followed by Bonferroni's multiple comparison test (C). Significance is indicated by * p < 0.05; ** p < 0.01; and **** p <0.0001.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Infection, Virus, Staining, Expressing, Two Tailed Test, Comparison

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Generation of BM CD8 + T RM cells does not require local infection or antigen presentation. (A) Percentage of CD69 + CD62L − cells within Influenza NP 366‐374 ‐specific CD44 + CD8 + T cells in spleen and BM 33 days after intranasal infection with Influenza A/HKx31. Data are shown from one representative experiment with n = 5 mice, out of two independent experiments; (B) Naïve WT mice were epicutaneously infected with HSV‐1 one day after i.v. transfer of 5 × 10 4 gBT‐I CD8 + T cells, which recognize the HSV‐1 K b ‐gB 498‐505 epitope and are identified as Vα2 + Thy1.1 + . Expression of CD69 was analyzed on donor cells in spleen and BM 40 days after infection; (C) Percentage of CD69 + within gBT‐I CD8 + T cells in spleen and BM, 11 months after in vitro activation and transfer; for (B) and (C), experiments were performed twice. (D) Frequency of gBT‐I cells within total CD8 α + cells in the spleen (left) or CD69 + within gBT‐I cells in the BM (right) of conjoined mice that received gBT‐I and were subsequently infected with HSV, as shown in (B); (E) Frequency of gBT‐I cells within total CD8 α + cells in the spleen (left) or CD69 + within gBT‐I cells in the BM (right) of conjoined mice that received in vitro activated gBT‐I cells, as shown in (C). For (D) and (E), organs were analyzed 3 weeks after conjoining. For (D) and (E), data are shown from one experiment with n = 3 mice. Data were analyzed by two‐tailed t ‐test, with matching. Significance is indicated by * p < 0.05 and ** p < 0.01.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Infection, Immunopeptidomics, Expressing, In Vitro, Activation Assay, Two Tailed Test

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Virus‐specific resident‐like CD8 + T cells are found within human BM. (A) Representative staining for CD69 and CD103 on CD8 + CD3 + T cells in human BM; (B and C) Expression of chemokine receptors CXCR6, CCR5, and CX 3 CR1 on CD69 − and CD69 + CD8 + T cells in human BM; (B) Representative histograms for CD69 − and CD69 + CD8 + T cells; (C) Expression levels depicted as geoMFI for the same populations. For (A–C), data are shown for n = 4 non‐HLA typed BM samples from four different donors; (D) Combinatorial coding analysis of human CD8 + BM T cells specific for CMV or EBV. Dually labelled MHC‐I tetramers loaded with the same immunodominant peptide from either CMV‐VTE, CMV‐TPR, or EBV‐RAK identify virus‐specific CD8 + T cells from BM of HLA‐typed donor #1 (percentages of specific cells is indicated in the gate); (E–G) Quantification of CMV‐ or EBV‐specific CD8 T cells and the percentage of CD69 + cells therein, in human BM. Data are shown for n = 5 HLA‐typed BM samples from five different donors; (E) Table describes detailed information on each specific T cell population detected; (F) The percentage of Tet+ cells detected within total CD8 + cells. For (F) and (G), results are shown as average ± SD. In panel (G), only those samples are indicated for which >100 CD69 + tetramer + CD8 + T cells were acquired. Data was analyzed by one‐way ANOVA followed by Tukey's multiple comparisons test (G) and two‐way ANOVA, with matching, followed by Bonferroni's multiple comparison test (C). Significance is indicated by * p < 0.05 and ** p < 0.01.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Virus, Staining, Expressing, Comparison

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Murine steady‐state BM lodges CD8 + T cell memory‐like cells with a resident phenotype. (A–D) Analysis of the CD8 + T cell memory‐like compartment in adult mice under SPF breeding. Data are shown for 1 experiment with n = 5 mice; (A) Frequency of memory CD44 + cells within CD8 αβ + TCR β + T cells in peripheral blood (PB), spleen, and BM; (B) Frequency of CD69 + cells of all CD44 + CD8 αβ + TCR β + T cells; (C) Representative FACS plots showing expression of CD62L and CD69 on memory CD8 T cells (defined as CD44 + CD8 αβ + TCR β + cells) in the different organs; (D) Frequency of memory CD8 + T cells subsets in spleen and BM, as gated for in panel C; (E–H) Relative mRNA expression of (E) Ccr7 , (F) Klf2 , (G) S1pr1 , and (H) Hobit relative to the housekeeping gene Cyclophilin A in sort‐purified memory CD8 + T cell subsets from BM, as defined in (C) and (D). Data is shown for four independent sorting experiments, each with pooled BM from n = 4 mice; (I) Heatmap of RNA sequencing data for the 30 genes that compose the universal transcriptional signature of lymphocyte tissue residency in CD8 + T NV (CD44 − CD62L + ), T CM (CD44 + CD62L + ), T EM (CD44 + CD62L − CD69 − ), and T RM (CD44 + CD62L − CD69 + ) cells that were sort‐purified from BM of naïve mice. Data are shown from three independent sorting experiments, each with pooled BM from n = 6 mice. Relative expression levels ( Z ‐scores) of genes are shown, color coded according to legend. Data are shown as mean ± SD (A and B) or mean ± SEM (E–H). Statistical analysis was performed with one‐way ANOVA followed by Tukey's multiple comparisons test (A and B) and two‐way ANOVA followed by Bonferroni's multiple comparison test (D–H). Significance is indicated by * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Expressing, Purification, RNA Sequencing, Comparison

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Specific features of BM CD8 + T RM cells. (A–D) Analysis of RNA sequencing data obtained for CD8 + T NV (CD44 − CD62L + ), T CM (CD44 + CD62L + ), T EM (CD44 + CD62L − CD69 − ), and T RM (CD44 + CD62L − CD69 + ) cells that were sort‐purified from BM of naïve mice. Data are shown from three independent sorting experiments, each with pooled BM from n = 6 mice; (A) Number of differentially expressed (DE) genes (downregulated in white, upregulated in black) between T RM versus T EM , T RM versus T CM , and T RM versus T NV . DE expressed genes were specifically up‐ or downregulated in BM T RM in comparison to their circulating counterparts, as determined by RNA sequencing (fold change [FC] > 1.5; and reads per kilobase per million mapped reads (RPKM) > 8]; (B) Venn diagram showing the overlap between genes DE by T RM and T EM , T RM , and T CM cells; (C) Heatmap displays relative amounts of transcripts of the 39 DE genes that were shared in the comparison shown in (B). Relative expression levels ( Z ‐scores) of genes are shown, color coded according to legend; (D) Correlation of gene ontology (GO)‐terms enriched in the sum of DE expressed genes between T RM versus T EM and T RM versus T CM (547 DE genes, FC > 1.5; RPKM = 8). GO‐terms were filtered to contain between 20 and 100 genes. Enrichment analysis revealed enrichment of the DE genes in 104 GO‐terms, which clustered into nine groups by comparison of their overlap index. Results are shown as a heatmap of the overlap indices, with the clustering and a color representation of each group on the left and a representative group name on the right; (E and F) Transcript levels of IFN‐γ in T CM , T EM , and T RM sorted from steady‐state mice, analyzed via RNAseq (E, obtained as in A–D) or RT‐qPCR (F, data are from four different sorting experiments, each with pooled BM from n = 4 mice); (G–I) Production of IFN‐γ by T CM , T EM , or T RM CD8 + T cells from LCMV‐infected mice. BM cells were incubated for 5 h with Brefeldin A in the absence (no stim.) or presence of GP 33‐41 peptide and IFN‐γ production was evaluated by ICS in the different memory populations. Data are shown for one representative experiment with n = 4 mice, out of two independent experiments; (G) Representative plots and quantification of the production of IFN‐γ in the absence (H) or presence of LCMV peptide (I); (J and K) Polyfunctionality of T EM and T RM cells was analyzed as in panels (G–I). Data are from one representative experiment with n = 5 mice, out of two independent experiments; (J) Co‐production of IFN‐γ and CCL3 by T EM or T RM CD8 + T cells from LCMV‐infected mice. Representative plots are shown; (K and L) Representative plots (K) and quantification (L) of T EM and T RM capable of simultaneously producing two, three, or four out of four cytokines (IFN‐γ, CCL3, IL‐2, and TNF‐α). Results in (H), (I), and (L) show mean ± SD. Statistical analysis was performed by one‐way ANOVA followed by Tukey's multiple comparisons test. Significance is indicated by ** p < 0.01 and *** p < 0.001.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: RNA Sequencing, Purification, Comparison, Expressing, Quantitative RT-PCR, Infection, Incubation

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: BM CD8 + T RM cells reside in the parenchyma in close contact with the circulation. (A and B) Comparison of DE genes between T RM from different organs. RNA sequencing data for BM T RM was acquired as in Fig. I, while data for liver, SI, and skin T RM were obtained from ref. ; (A) Venn diagram showing the overlap between DE genes by T RM from BM, Liver, SI, and skin, compared to splenic T NV (Sp) (FC > 1.5; RPKM = 8); (B) Correlation plot depicting the similarity between T RM in different organs. The correlation is pairwise, and was calculated using log RPKM values of the 136 DE genes that were significant in all four contrasts; (C–E) Intravascular staining to probe the localization of T RM within the BM. Mice were i.v. injected with 3 μg of an antibody against CD8 a . Organs were obtained 2 min after injection and processed immediately. Data are shown for one representative experiment with n = 3 mice, out of two independent experiments; (C) A representative histogram comparing the staining with the anti‐CD8 α antibody in CD8 β + CD44 + cells from spleen, peripheral blood (PB), liver, and BM; geoMFI of CD8 α staining in (D) CD8 β + CD44 + cells from the different organs or (E) CD8 β + memory subsets within the BM; (F and G) Immunofluorescence analysis of (F) CD8 α + cells or (G) CD69 + CD8 α + cells within steady‐state BM. Scale bar = 50 μm. Vasculature was visualized with antibodies against CD31 and CD144 and nuclei with Helix NP green staining. One representative region from a tile scan of 4 × 7 images is shown. The full tile scan is shown in Supporting Information Fig. ; 40× magnification. Each CD8 a + cell is encircled in white to identify these cells across the different stainings. CD69 + CD8 α + cells are marked with an arrow.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Comparison, RNA Sequencing, Staining, Injection, Immunofluorescence

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: Maintenance of BM CD8 + T RM cells depends on IL‐15 and Hobit. (A–C) In vitro‐activated gBT‐I cells were transferred into WT or IL‐15 −/− mice and, after 30 days, the presence of total gBT‐I and CD69 + gBT‐I was analyzed in the BM. Results are shown as (A) representative plots, (B) quantification of the % of gBT‐I within total CD8 α + cells and (C) quantification of TRM (CD69 + ) cells within gBT‐I cells in the BM, from one experiment with n = 5 WT and 4 IL‐15 −/− mice. Results show mean ± SD; experiment was performed twice. (D and E) WT (Blimp +/+ Hobit +/+ ) or mice deficient for Hobit (Blimp +/+ Hobit −/− ), Blimp1 (Blimp −/‐ Hobit +/+ ), or both (Blimp −/− Hobit −/− ) were infected i.n. with Influenza A/HKx31. BM was harvested and analyzed 63 dpi. Results are shown from one representative experiment with n = 5 mice, out of three independent experiments, as (D) percentage of D b ‐NP 366‐374 + cells within total CD8 αβ + T cells and (E) percentage of T RM (CD69 + CD62L − ) cells within D b ‐NP 366‐374 + CD8 αβ + T cells. Data were analyzed by two‐tailed t ‐test (B and C) or one‐way ANOVA followed by Tukey's multiple comparisons test (D and E). Significance is indicated by * p < 0.05 and ** p < 0.01.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: In Vitro, Infection, Two Tailed Test

Journal: European Journal of Immunology

Article Title: Peripheral and systemic antigens elicit an expandable pool of resident memory CD8 + T cells in the bone marrow

doi: 10.1002/eji.201848003

Figure Lengend Snippet: The pool of BM CD8 + T RM cells is expandable. (A–C) Distribution of CD8 αβ + memory populations specific for mCMV acute and inflationary epitopes analyzed 65 days p.i.; Representative plots are shown for (A) acute epitopes M45 and M57 and (B) inflationary epitopes M38 and m139. Tetramer stainings are shown in the left panels and the gating for T RM (CD62L − CD69 + ) and T EM (CD62L − CD69 − ) is shown in the right panels; (C) Percentage of T EM and T RM within live BM cells for each mCMV‐specific population. Data is shown for one representative experiment with n = 4 mice, out of two independent experiments; (D–F) Distribution of OT‐I memory populations analyzed more than 30 days after the last immunization in mice that received one or three homologous boosts to generate OVA‐specific memory. Data are shown for one experiment with n = 5 mice that received one challenge and 4 mice that received three challenges; (D) Schematic depiction of the experimental setup; (E) Percentage of OT‐I cells within total CD8 α + cells in the BM; (F) Percentage of T CM , T EM , and T RM within OT‐I cells in the BM after one or three homologous challenges; (G–I) Distribution of OVA‐specific CD8 αβ + memory populations analyzed more than 60 days after the first challenge in mice that received only Lm‐OVA or Lm‐OVA followed by LCMV Armstrong infection, more than 30 days after the first challenge. Data are shown for one experiment with n = 5 mice/group. Results from the reverse experiment are shown in Supporting Information Fig. ; (G) Schematic depiction of the experimental setup; (H) Percentage of K b ‐OVA 257‐264 + T RM cells of live BM cells; (I) Percentage of total T RM within live BM cells. Data were analyzed by two‐tailed t ‐test (E, H, and I) and two‐way ANOVA, followed by Bonferroni's multiple comparison test (C and F). Significance is indicated by ** p < 0.01 and *** p < 0.001.

Article Snippet: The following antibodies were used: CD8α‐Alexa Fluor 647 (53‐6.7, Biolegend), CD144/VE‐cadherin biotin (BV13, Biolegend), CD31/PECAM‐1 biotin (MEC 13.3, BD Pharmingen), CD69 (R&D systems), Donkey‐Anti‐Goat IgG Alexa Fluor 568 (Invitrogen), and Streptavidin eFluor 450 (eBioscience).

Techniques: Infection, Two Tailed Test, Comparison

Journal: Cancer Research Communications

Article Title: Immuno-PET Imaging of CD69 Visualizes T-Cell Activation and Predicts Survival Following Immunotherapy in Murine Glioblastoma

doi: 10.1158/2767-9764.CRC-22-0434

Figure Lengend Snippet: Kinetics of activation markers on T cells following ICI treatment. Mice with intracranial GL261 gliomas were treated with ICI, and analyzed at the designated time points after treatment, as in . A, Percentages of CD69 + of CD8 + (left) and CD4 + TILs (right). B, Percentages of CD69 + of CD8 + (left) and CD4 + T cells (right) in spleen. C, Trajectories of CD69 + /CD8 + in TILs (green line) and spleen (purple line) ratios at the designated time point compared with the pretreatment group. D, Percentages of PD-1 + of CD8 + (left) and CD4 + TILs (right). E, Percentages of PD-1 + of CD8 + (left) and CD4 + T cells (right) in spleen. F, Trajectories of PD-1 + /CD8 + in TILs (blue line) and spleen (red line) ratios at the designated time point compared with the pretreatment group. G, Percentages of TIM3 + of CD69 + /CD8 + TILs. For A, B, D, E, and G, mean ± SEM values are shown. Scattered dots represent individual mice. Shown are representative data of two independent experiments. n = 4 per group. One-way ANOVA with multiple comparison test (*, P < 0.05; **, P <0.01; ***, P <0.001). C and F, Two-way ANOVA with multiple comparison test is used to compare the ratios between TILs and spleen T cells at each time point (*, P < 0.05; **, P <0.01; ***, P <0.001).

Article Snippet: In the next step, all wells were washed with washing buffer, and different concentrations of anti-CD69 [clone CD69.2.2 (BioXcell) or clone H1.2F3 (Invitrogen)] were added to the wells and incubated for 2 hours at room temperature with shaking.

Techniques: Activation Assay

Journal: Cancer Research Communications

Article Title: Immuno-PET Imaging of CD69 Visualizes T-Cell Activation and Predicts Survival Following Immunotherapy in Murine Glioblastoma

doi: 10.1158/2767-9764.CRC-22-0434

Figure Lengend Snippet: CD69 expression increases on TILs following ICI treatment in patients with GBM. A–C, scRNA-seq analysis of sorted CD45 + immune cells ( n = 156,766 cells) from 40 patients with GBM: 14 GBM.new, 12 GBM.rec, and 14 GBM.pembro. A, UMAP cell clustering analysis of tumor cell cluster (SOX4; top right), myeloid cell cluster (CD14; middle right), and lymphocyte cluster (CD3D; bottom right). B, UMAP data color-coded by the expression of CD3 (red), CD69 (purple; top), and coexpression (merge) in each group of patients (bottom). C, Dot plot analysis of cells within the lymphocyte cluster; the size of the dots corresponds to the percentage of cells expressing the feature (gene). The color represents the average expression level.

Article Snippet: In the next step, all wells were washed with washing buffer, and different concentrations of anti-CD69 [clone CD69.2.2 (BioXcell) or clone H1.2F3 (Invitrogen)] were added to the wells and incubated for 2 hours at room temperature with shaking.

Techniques: Expressing

Journal: Cancer Research Communications

Article Title: Immuno-PET Imaging of CD69 Visualizes T-Cell Activation and Predicts Survival Following Immunotherapy in Murine Glioblastoma

doi: 10.1158/2767-9764.CRC-22-0434

Figure Lengend Snippet: Immuno-PET of 89 Zr-DFO-CD69 Ab visualizes the TME after ICI treatment in a GBM mouse model. Mice were inoculated with GL261 cells, and evaluated by immuno-PET of 89 Zr-DFO-CD69 Ab, comparing the treatment (ICI) group to the control group (represented by orange and blue dots, respectively). A, Schematic showing timeline of tumor inoculation, ICI treatment, tail vein injection of 89 Zr-DFO-CD69 Ab, immuno-PET, and BioD. B, Representative coronal head images from immuno-PET of 89 Zr-DFO-CD69 Ab of ICI-treated and control mice at designated time points. Scales show SUVs of PET (SUV; colored) and CT (HU; gray). C and D, Comparison between ICI-treated mice and control at designated time points of tumor-specific regions’ SUV max (C) and SUV max TBR (D). E, Representative coronal-ventral and sagittal 3D whole-body MIP PET images of 89 Zr-DFO-CD69 Ab of control and ICI-treated mice acquired 6 days after tracer administration. The scale shows SUVs of PET. F, Day 6 BioD results of blood- and tumor site–associated radioactivity, assessed as %ID/g. In C, D, and F, bars show means ± SEM. Scattered dots represent individual mice. Shown are representative data of three independent experiments, n = 5 per group. C and D, Multiple unpaired t test with Welch correction. F, Two-way ANOVA with multiple comparison test (*, P < 0.05; **, P <0.01; ***, P <0.001). IP, intraperitoneal.

Article Snippet: In the next step, all wells were washed with washing buffer, and different concentrations of anti-CD69 [clone CD69.2.2 (BioXcell) or clone H1.2F3 (Invitrogen)] were added to the wells and incubated for 2 hours at room temperature with shaking.

Techniques: Injection, Radioactivity

Journal: Cancer Research Communications

Article Title: Immuno-PET Imaging of CD69 Visualizes T-Cell Activation and Predicts Survival Following Immunotherapy in Murine Glioblastoma

doi: 10.1158/2767-9764.CRC-22-0434

Figure Lengend Snippet: Immuno-PET of 89 Zr-DFO-CD69 Ab as a prognostic predictor after ICI treatment in a GBM mouse model. Survival follow-up of mice inoculated with GL261 cells, and evaluated by immuno-PET of 89 Zr-DFO-CD69 Ab, comparing treatment (ICI) group to control group (represented by orange and blue dots, respectively). A, Schematic showing timeline of tumor inoculation, ICI treatment, MRI, tail vein injection of 89 Zr-DFO-CD69 Ab, immuno-PET, and survival follow-up. B, The overall survival (OS) rates of the ICI-treated group and control group are plotted using Kaplan–Meier survival curves, with log-rank (Mantel–Cox) curve comparison test. C, Right, heat map display of Pearson correlation coefficient analysis between immuno-PET signals and survival in the two groups. Results are shown for each SUV measurement method (SUV max , SUV max TBR, SUV mean , and SUV mean TBR) in each immuno-PET designated time point (days 1, 2, 3, 4, and 6). Shown are representative data of two independent experiments, n = 4–5 mice per group (*, P < 0.05; **, P <0.01). Left, examples of scatter diagrams of Pearson correlation coefficient taken from immuno-PETs on day 2 of SUV max and SUV max TBR. Scattered dots represent individual mice, n = 4–5 mice per group.

Article Snippet: In the next step, all wells were washed with washing buffer, and different concentrations of anti-CD69 [clone CD69.2.2 (BioXcell) or clone H1.2F3 (Invitrogen)] were added to the wells and incubated for 2 hours at room temperature with shaking.

Techniques: Injection

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Different Triple-Negative Breast Cancer Tumor Cell Lysates (TCLs) Induce Discrepant Anti-Tumor Immunity by PD1/PDL-1 Interaction

doi: 10.12659/MSM.911689

Figure Lengend Snippet: CD69 detection of PBMC stimulated with TNBC TCL. PBMC were cultured for 48 h in medium containing P65-1187TCL, P65-231TCL, P65-1937TCL, P65-436TCL, or 1×PBS. The expression of CD69 in PBMCs was determined by flow cytometry. Number in each histogram represents the CD69 mean fluorescence intensity of the sample.

Article Snippet: Thereafter, the cells were stained with fluorescein isothiocyanate-labeled anti-CD69 (QED Bioscience; San Diego, CA, USA) to detect the activation of PBMC or stained with PE-labeled anti-IFN-γ and fluorescein isothiocyanate-labeled anti-CD8 antibody (Kaiji Biotechnology Co., Nanjing, China) to analyze CTL.

Techniques: Cell Culture, Expressing, Flow Cytometry, Fluorescence