Noninvasive imaging of immune responses - PubMed (original) (raw)

Noninvasive imaging of immune responses

Mohammad Rashidian et al. Proc Natl Acad Sci U S A. 2015.

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Abstract

At their margins, tumors often contain neutrophils, dendritic cells, and activated macrophages, which express class II MHC and CD11b products. The interplay between stromal cells, tumor cells, and migratory cells such as lymphocytes creates opportunities for noninvasive imaging of immune responses. We developed alpaca-derived antibody fragments specific for mouse class II MHC and CD11b products, expressed on the surface of a variety of myeloid cells. We validated these reagents by flow cytometry and two-photon microscopy to obtain images at cellular resolution. To enable noninvasive imaging of the targeted cell populations, we developed a method to site-specifically label VHHs [the variable domain (VH) of a camelid heavy-chain only antibody] with (18)F or (64)Cu. Radiolabeled VHHs rapidly cleared the circulation (t1/2 ≈ 20 min) and clearly visualized lymphoid organs. We used VHHs to explore the possibility of imaging inflammation in both xenogeneic and syngeneic tumor models, which resulted in detection of tumors with remarkable specificity. We also imaged the infiltration of myeloid cells upon injection of complete Freund's adjuvant. Both anti-class II MHC and anti-CD11b VHHs detected inflammation with excellent specificity. Given the ease of manufacture and labeling of VHHs, we believe that this method could transform the manner in which antitumor responses and/or infectious events may be tracked.

Keywords: PET imaging; camelid single domain antibodies; cancer; inflammation; non-invasive imaging.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

VHH7 (anti-mouse class II MHC) and VHHDC13 (anti-mouse CD11b) stain secondary lymphoid organs. VHHs were site-specifically labeled with Texas Red or Alexa 647 via sortagging. In A_–_F, images were acquired using two-photon microscopy. In A, B, and C, the Left and Middle panels show the Texas Red and GFP channels, respectively. The Right panels are overlays. (A) Images of a lymph node of a class II MHC-eGFP knock-in mouse, no VHH injection prior to imaging. (B) Images of a lymph node of a class II MHC-eGFP knock-in mouse injected with 20 μg of VHH7-Texas Red 90 min prior to imaging. (C) Images of a lymph node of a class II MHC-/- mouse injected with 20 μg of VHH7-Texas Red 90 min prior to imaging. (D) Image of a lymph node of a B6 mouse injected with 20 μg of VHH7-Texas Red 90 min prior to imaging. (E1 and E2) Image of a lymph node of a B6 mouse injected with 20 μg of VHHDC13-Texas Red 90 min prior to imaging. E1 is a larger magnification of the lymph node shown in E2. (F) Image of a lymph node of a B6 mouse with no VHH injected. All injections were done intravenously. See Fig. S1 for images of spleen. See

Images S1–S4

for high-resolution images of lymph nodes. (G) WT or class II MHC-deficient mice were injected or not with 2 or 20 μg of VHH7-Alexa 647. After 2 h, spleen and brachial lymph nodes (bLNs) were harvested, and cell suspensions were stained with anti-CD19 and anti-CD3 antibodies prior to FACS analysis. (H) WT or CD11b-deficient mice were injected or not with 2 or 20 μg of VHHDC13-Alexa 647. After 2 h, spleen and brachial lymph nodes (bLNs) were harvested, and cell suspensions were stained with anti-CD11b and anti-CD3 antibodies prior to FACS analysis. Histograms are representative of three to four mice with similar results.

Fig. 2.

Fig. 2.

(A_–_E) Site-specific 18F or 64Cu labeling of single-domain antibodies (VHHs) using sortase. (A) A single-domain antibody fragment (VHH), equipped at its C terminus with the LPXTG sortase recognition motif followed by a His tag, is incubated with sortase A, which cleaves the threonine–glycine bond to yield the reactive thioacyl intermediate. Addition of a peptide with N-terminal glycine residues and a functional moiety of choice enables site-specific modification of the VHH. We thus modified a VHH with a (Gly)3-tetrazine (Tz), as confirmed by LC-MS (liquid chromatography-mass spectrometry) (D, VHH7-Tz). (B) A tosyl-TCO and 18F-K222/K2CO3 were combined to produce 18F-TCO. (C) 18F-TCO was added to the Tz-modified VHH, and, after ∼20 min, the labeled VHH was retrieved by rapid size-exclusion chromatography. (E) The sortase reaction was used to install a NOTA functionality at the C terminus of a VHH followed by addition of 64Cu2+ to produce 64Cu-VHH. (F_–_M) 18F-VHH7 (anti-mouse class II MHC) detects secondary lymphoid organs. (F and G) PET-CT images of class II MHC-/- (G) and C57BL/6 (F) mice 2 h postinjection of 18F-VHH7; numbers indicate (i) lymph nodes (numbers 1, 2, 3, 4, and 7), (ii) thymus (number 5), and (iii) spleen (number 6). (H and I) Coronal PET-CT images of C57BL/6 mouse imaged with 18F-VHH7, moving from anterior to posterior. In H and I, different sets of lymph nodes and thymus are visible. (K and L) Coronal PET-CT images of an MHC-II-/- mouse imaged with 18F-VHH7, moving from anterior to posterior. In neither K nor L, lymph nodes or thymus is visible. (J and M) PET-CT as maximum intensity projections of all slices for a C57BL/6 and a class II MHC-/- mouse 2 h postinjection of 18F-VHH7. In green, accumulation of 18F-VHH7 in lymph nodes and thymus. (PET scale bars have arbitrary units.) See Movies S1_A_, S1_B_, S2_A_, and S2_B_ for 3D visualization of secondary lymphoid organs. (N and O) PET signals in vivo and postmortem biodistribution in all organs.

Fig. 3.

Fig. 3.

18F-VHH7 (anti-mouse class II MHC) and 18F-VHHDC13 (anti-mouse CD11b) detects inflammation. Tumor-associated class II MHC+ cells were visualized using 18F-VHH7. A NOD-SCID mouse was inoculated subcutaneously on the back of the left shoulder with 5 × 106 human Mel-Juso melanoma cells and imaged 35 days postinjection. (A_–_C) Coronal PET-CT images, moving from anterior to posterior. In A and B, different sets of lymph nodes are visible. In C, tumor-associated class II MHC+ cells are visible, attributable to influx of host-derived class II MHC+ cells. (D) PET-CT as maximum intensity projections of all slices. See Movie S3 for a 3D visualization of lymph nodes and tumor-associated class II MHC+ cells. (E) 18F-VHH7 detects class II MHC+ cells associated with small tumors at early stages. NOD-SCID mice were inoculated with Mel-Juso cells as in A, 20 d (Upper) and 6 d (Lower) prior to imaging. Transverse PET and CT images (Left and Right, respectively) are shown for better visualization of the class II MHC+ cells at the site of cancer cells. Images are all window-leveled to the same intensity. Tumors and associated class II MHC+ cells are highlighted with arrows. Axillary (label A), brachial (label B), and mediastinal (label M) lymph nodes and thymus (label T) are shown in E, Lower. (F and G) A NOD-SCID mouse was inoculated subcutaneously on the back of the left shoulder with 5 × 106 human Mel-Juso melanoma cells and imaged 35 days postinjection with 18F-VHHDC13. Tumor-associated CD11b+ cells are visible, attributable to influx of host-derived CD11b+ cells. See Movie S4 for a 3D visualization. (H and I) FACS analysis of tumor-infiltrating immune cells. The next day, tumors were excised and digested with collagenase D, and tumor-infiltrating cells were obtained after Percoll gradient. Cell suspensions were then stained for FACS analysis. (H) Histograms show the FACS staining of mouse CD45+ tumor-infiltrating cells with VHH7-Alexa 647. Histograms on the Left are gated on CD11c+CD11b+ cells (dendritic cells), and histograms on the Right are gated on CD11c−CD11b+cells (macrophages and other myeloid cells) for the indicated time points. (I) Tumor-infiltrating cells were harvested 35 d after tumor inoculation as in H and stained with VHHDC13-Alexa 647. Histograms show the levels of CD11b as measured by VHHDC13 on the indicated cell populations. Spleen from the same mouse is shown for comparison. Histograms are representative of two to four mice with similar results. (J) PET signals in vivo for the indicated experiment in D and F. (K) PET signals in vivo for the indicated experiment in E. (L) Hematoxylin/eosin (H&E) stains of the tumor from the mice in the part D (human melanoma tumor in NOD-SCID mouse; 35 d postinjection of the cells).

Fig. 4.

Fig. 4.

18F-VHH7 (anti-mouse class II MHC) and 18F-VHHDC13 (anti-mouse CD11b) detects inflammation. (A_–_C) Tumor-associated CD11b+ and class II MHC+ cells were visualized using 18F-VHHDC13 and VHH7, with 18F-FDG failing to yield a strong tumor-specific signal. Mice were inoculated subcutaneously on the back of the left shoulder with 1 × 106 B16 melanoma cells and imaged 7 d postinjection. See Movies S9, S10, and S11 for a 3D visualization. (A) 18F-VHHDC13 was injected 90 min prior to imaging. The tumor on the left shoulder is clearly visible, attributable to the influx of CD11b+ cells. (B) 18F-VHH7 was injected 90 min prior to imaging. The lymph nodes and the tumor on the left shoulder are visible, attributable to the influx of class II MHC+ cells; detection of influx of CD11b+ cells is superior to the detection of influx of class II MHC+ cells. (C) 18F-FDG was injected 90 min prior to imaging. The tumor could be visualized via 18F-FDG with lower specificity compared with VHHs specifically relative to anti-CD11b. (D_–_F) Complete Freund’s adjuvant (CFA) was injected into the left paw of C57BL/6 mice, and 18F-VHHDC13 (D), 18F-VHH7 (E), or 18F-FDG (F) was used 24 h after CFA injection. PET-CT images were obtained 1.5 h postinjection of 18F agents. Images are all window-leveled to the same intensity for comparison. See Movies S5_A_, S5_B_, S5_C_, S6_A_, S6_B_, S7, S8_A_, and S8_B_ for a 3D visualization. (PET scale bars have arbitrary units.) (G) WT or the indicated KO mice were injected subcutaneously with 8 × 105 B16 melanoma cells, and 8 days later injected i.v. with 20 μg of the indicated VHH conjugated to Alexa 647. After 90 min of VHH injection, animals were sacrificed, spleen and tumor were excised, and single-cell suspensions were further stained with anti-CD3, anti-CD19, anti-CD11b, and anti-CD45 for FACS analysis. Histograms are representative of two to four mice with similar results. (H) PET signals in vivo for the indicated experiments in A_–_C. (I) PET signals in vivo for the indicated experiments in D_–_F.

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