c57bl 6 mouse serum  (Innovative Research Inc)

Journal: Nature methods

Article Title: In vivo detection of antigen-specific CD8 T cells by immuno-positron emission tomography

doi: 10.1038/s41592-020-0934-5

Figure Lengend Snippet: Analysis of synTac specificity in vitro . Splenocytes from C57BL/6 mice treated with VHH CD11b -E7 (shortened as VHH-E7) plus adjuvant or from IAV-infected mice were incubated overnight with HPV E7 49–57 (RAHYNIVTF), HPV E7 synTac, IAV NP 366–374 (ASNENMETM), or IAV NP synTac (100 nM equivalent). The number of IFN-γ-secreting cells was measured via ELISpot assay. Shown are means ± SD of an experiment performed in quadruplicate (biological replicates and is representative of two independent experiments (**** p < 0.0001; two-sided Student’s t-test).

Article Snippet: 60 μg of TMR-conjugated synTac was incubated in C57BL/6 serum (501003574, Innovative Research Inc.) at 37°C for the desired time, then the mixture was loaded into a 100 μL loop and run on an analytical gel filtration column (Superose 6 Increase 10/300 GL, GE Life Sciences).

Techniques: In Vitro, Infection, Incubation, Enzyme-linked Immunospot

Journal: Nature methods

Article Title: In vivo detection of antigen-specific CD8 T cells by immuno-positron emission tomography

doi: 10.1038/s41592-020-0934-5

Figure Lengend Snippet: ELISpot analysis of HPV E7-specific T cells in (shortened as VHH-E7 above) treated mice and detection of CD8 T cells in HPV E7-expressing tumors by VHH-based immunoPET. Wild-type C57BL/6 mice were challenged with 3×10 5 C3.43 cells. When tumors were palpable (~14 days later), mice were treated IP with adjuvant (adj. = 50 μg Poly(I:C) + 50 μg agonistic anti-CD40 Ab) or VHH CD11b -E7 plus adjuvant (n = 3/group). 8 days after treatment, spleens and tumors were harvested and E7 49–57 -specific CD8 T cells were enumerated via IFN-γ ELISpot. A) Representative examples of quadruplicate wells of the ELISpot assay. B) Quantification of IFN-γ secreting cells detected in the spleens (left) and C3.43 tumors (right) of the treated mice (means ± SD are shown; n = 3; *** p < 0.001; two-sided Student’s t-test). C) C3.43 tumor-bearing mice from (B) were retro-orbitally injected with an anti-CD8 VHH labeled with 89 Zr 7 days after treatment and imaged the following day by PET-CT prior to spleen and tumor resection. Representative PET-CT images are shown. White arrows in the maximal intensity projection (MIP) coronal (i) and transverse (ii) images indicate the sites of the tumors.

Article Snippet: 60 μg of TMR-conjugated synTac was incubated in C57BL/6 serum (501003574, Innovative Research Inc.) at 37°C for the desired time, then the mixture was loaded into a 100 μL loop and run on an analytical gel filtration column (Superose 6 Increase 10/300 GL, GE Life Sciences).

Techniques: Enzyme-linked Immunospot, Expressing, Injection, Labeling, Positron Emission Tomography-Computed Tomography

Journal: Nature methods

Article Title: In vivo detection of antigen-specific CD8 T cells by immuno-positron emission tomography

doi: 10.1038/s41592-020-0934-5

Figure Lengend Snippet: PET-CT imaging with the HPV E7 synTac. A-C) C3.43 tumor-bearing C57BL/6 mice were either treated with adjuvant only (A) or VHH CD11b -E7 (shortened as VHH-E7) plus adjuvant (B-C). ~50 μCi (1850 kBq) 64 Cu-labeled HPV E7 (A-B) or LCMV P14 (C) synTac was injected retro-orbitally 7 days after treatment, and PET-CT scanning was performed the next day (8 days after treatment). Representative MIP, coronal, and transverse (cross-section of tumor) PET-CT images are shown with close-up images of the transverse tumor sections in which PET signal is only seen in the tumors of VHH CD11b -E7 treated mice imaged with the 64 Cu-labeled HPV E7 synTac (B). White arrows indicate the location of the tumors. D) Quantification of C3.43 intratumoral PET signals over background signal (hindleg muscle) as observed in mice from (A-C) (means ± SD are shown; n = 4/group; *p < 0.05; two-sided Student’s t-test). E-F) C3.43 and B16 co-tumor-bearing mice were treated with VHH CD11b -E7 plus adjuvant. 64 Cu-labeled HPV E7 synTac was injected 7 days after treatment, and PET-CT scanning was performed the next day (8 days after treatment). Representative coronal and transverse PET-CT images are shown with close-up images of the tumors. A C3.43 (E; white arrows) and B16 tumor (F; white arrows) are shown separately from the same mouse. PET signal was greater in C3.43 tumors that expressed the appropriate HPV E7 antigen than that of B16 tumors in the same animal. G) Quantification of PET signals observed in C3.43 tumors over those observed in B16 tumors of mice treated with adjuvant only or VHH CD11b -E7 plus adjuvant (means ± SD are shown; n = 4/group; **p < 0.05; two-sided Student’s t-test).

Article Snippet: 60 μg of TMR-conjugated synTac was incubated in C57BL/6 serum (501003574, Innovative Research Inc.) at 37°C for the desired time, then the mixture was loaded into a 100 μL loop and run on an analytical gel filtration column (Superose 6 Increase 10/300 GL, GE Life Sciences).

Techniques: Positron Emission Tomography-Computed Tomography, Imaging, Labeling, Injection

Journal: Nature methods

Article Title: In vivo detection of antigen-specific CD8 T cells by immuno-positron emission tomography

doi: 10.1038/s41592-020-0934-5

Figure Lengend Snippet: PET-CT imaging with the IAV NP synTac. Non-tumor bearing C57BL/6 mice were infected with IAV via nasal drip 9 days prior to analysis of IAV NP-specific CD8 T cells. A-B) ELISpot analysis of IAV NP-specific CD8 T cell responses in the spleens (A) and lungs (B) of IAV-infected or uninfected control mice (means ± SD are shown; n = 3/group; ***p < 0.001; two-sided Student’s t-test). C-D) Coronal (C; full body top and close-up of lungs bottom) and transverse (D) PET-CT images of IAV-infected mice retro-orbitally injected with 64 Cu-labeled IAV NP synTac (left) or 64 Cu-labeled HPV E7 synTac (middle) 9 days after IAV infection. Uninfected control mice were imaged with the 64 Cu-labeled IAV NP synTac (right). E) Quantification of 64 Cu-labeled IAV NP synTac PET signal in the lungs of IAV-infected or control mice over background signal (hindleg muscle). Mice were injected with 64 Cu-labeled IAV NP synTac 8 days after IAV infection and PET-CT scanning was performed the next day (9 days after infection). IAV-infected mice were also given the 64 Cu-labeled HPV E7 synTac in which the PET signal in the lungs was similar to that observed in uninfected mice (means ± SD are shown; n = 3/group; *p <0.05). F) Quantification of synTac PET signals in the lungs before (black) and after (red) abdominal organ resection. The function of the red axis (right) is to show the value of the signal in the lungs after organ resection and coordinates with the red points in the graph. IAV-infected mice were injected with 64 Cu-labeled IAV NP or HPV E7 synTac 8 days after IAV infection and PET-CT imaging was performed the next day (9 days after infection). Uninfected control mice were given the 64 Cu-labeled IAV NP synTac (means ± SD are shown; n = 3/group; *p <0.05). G-I) PET-CT imaging with synTacs before (left) and after (right) abdominal organ resection. IAV-infected mice were injected with 64 Cu-labeled IAV NP synTac (G) or 64 Cu-labeled HPV E7 synTac (H) 8 days after IAV infection and PET-CT imaging was performed the next day (9 days after infection). I) Uninfected control mice were imaged with the 64 Cu-labeled IAV NP synTac. Shown are representative MIP PET-CT images of the same mice before (left) and after (right) abdominal organ resection (n = 3/group).

Article Snippet: 60 μg of TMR-conjugated synTac was incubated in C57BL/6 serum (501003574, Innovative Research Inc.) at 37°C for the desired time, then the mixture was loaded into a 100 μL loop and run on an analytical gel filtration column (Superose 6 Increase 10/300 GL, GE Life Sciences).

Techniques: Positron Emission Tomography-Computed Tomography, Imaging, Infection, Enzyme-linked Immunospot, Injection, Labeling

Journal: Nature methods

Article Title: In vivo detection of antigen-specific CD8 T cells by immuno-positron emission tomography

doi: 10.1038/s41592-020-0934-5

Figure Lengend Snippet: SynTac PET-CT imaging following different radio-labeling strategies. A) G 3 -NOTA-azide was attached to the HPV E7 synTac via sortase followed by the addition of a 20 kDa PEG (PEG 20 ) moiety (as DBCO-PEG 20 ) with a click reaction. PEGylation of the synTac was verified as an upward size-shift (~40 kDa as two PEG 20 moieties are attached per synTac; i.e., one per chain) following PAGE and Coomassie staining (right lane). B) C3.43 tumor-bearing mice were treated with VHH CD11b -E7 plus adjuvant. Mice were then retro-orbitally injected with 64 Cu-labeled HPV E7 synTac (left) or PEGylated 64 Cu-labeled HPV E7 synTac (right) 7 days after treatment, and PET-CT scanning was performed the next day (8 days after treatment). Representative MIP (i. whole body) and transverse (ii. cross-section of tumors; iii. cross-section of livers) PET-CT images are shown. C) PET-CT scanning was performed on wild-type C57BL/6 mice injected with HPV E7 synTac labeled with 64 Cu (imaged the following day), 18 F-FDG (imaged 4 hrs after injection), or 89 Zr (imaged the following day). Scaling for 64 Cu and 18 F-FDG are the same as in B and the 89 Zr image is scaled at 1.5–30 %ID/g. Representative coronal (top; whole body) and transverse (bottom; cross-section of livers) PET-CT images are shown. D) The IAV NP synTac was pre-treated with PNGase F resulting in deglycosylation of the Fc region as observed by a downward size-shift following SDS-PAGE and Coomassie staining (right lane). The bottom band in the right lane is PNGase F (~36 kDa). E) Mice were injected with PNGase F-digested 64 Cu-labeled IAV NP synTac 8 days after IAV infection and PET-CT imaging was performed the next day (9 days after infection) (left). Uninfected control mice were also imaged with the PNGase F-digested 64 Cu-labeled IAV NP synTac (right). Shown are representative coronal (top) and transverse (bottom; cross-section of lungs) PET-CT images of an IAV-infected and uninfected mouse. F) FcRn knock-out (KO) and wild-type C57BL/6 mice were injected with 64 Cu-labeled IAV NP synTac and PET-CT imaging was performed the next day. Representative MIP PET-CT images are shown. All experiments shown were performed at least three independent times with similar results.

Article Snippet: 60 μg of TMR-conjugated synTac was incubated in C57BL/6 serum (501003574, Innovative Research Inc.) at 37°C for the desired time, then the mixture was loaded into a 100 μL loop and run on an analytical gel filtration column (Superose 6 Increase 10/300 GL, GE Life Sciences).

Techniques: Positron Emission Tomography-Computed Tomography, Imaging, Labeling, Staining, Injection, SDS Page, Infection, Knock-Out