Abstract
Imaging inflammation holds immense potential for advancing the diagnosis, treatment and prognosis of many conditions1,2,3. The lack of a specific and sensitive positron emission tomography (PET) probe to detect inflammation is a critical challenge. To bridge this gap, we present CD45-PET imaging, which detects inflammation with exceptional sensitivity and clarity in several preclinical models. Notably, the intensity of the CD45-PET signal correlates robustly with the severity of disease in models of inflammatory lung and bowel diseases, outperforming 18F-fluorodeoxyglucose PET, the most widely used imaging modality for inflammation globally. Longitudinal CD45-PET imaging further enables precise monitoring of dynamic changes in tissue-specific inflammatory profiles. Finally, we developed a human CD45-PET probe for clinical translation that effectively detects human immune cells in a humanized mouse model. CD45-PET imaging holds substantial clinical promise, offering a tool for guiding diagnostic and therapeutic decisions for inflammatory diseases through a precise, whole-body assessment of the inflammation profiles of individual patients.
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Acknowledgements
We appreciate the support provided by the Flow Cytometry Core in the Department of Cancer Immunology, as well as the teams in Pathology, Research Operations, Radiation Safety and the Animal Facility at the Dana-Farber Cancer Institute and Boston Children’s Hospital. Their contributions greatly aided this project. We thank L. Clark, A. Day, C. Liddle, and E. McGondel for their support. We thank the Crimson Core at BWH Clinical Laboratories for their support in providing PBMCs. We thank H. Ploegh for his support throughout the project and our colleagues D. B. Rowley and M. Kircher for insightful discussions and guidance that were instrumental to advancing our work. We also thank J. Moslehi and his research team for their insightful comments and helpful discussions; and K. Wucherpfennig and K. Burns at the Dana-Farber Cancer Institute for their support and guidance throughout the project. This work was supported by the Dana-Farber Cancer Institute (M.R.), the Dana-Farber Cancer Institute Innovation Research Fund Award (M.R.), the Parker Institute for Cancer Immunotherapy (M.R.), the National Institutes of Health (NIH), National Cancer Institute K22-CA226040 (M.R.), R01-CA255216 (Hidde Ploegh and M.R.), the NIH, National Institute of Allergy and Infectious Disease (NIAID) R01-AI165666 (M.R.), the Harvard Medical School Office of Scholarly Engagement (J.E.R.) and the Khoury Innovation Award from the Brigham and Women’s Hospital Heart and Vascular Center (S.D.).
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Conceptualization and design: A.S.F., J.E.R. and M.R. Investigation: A.S.F., J.E.R., H.H.A., I.G.K., S.T., K.N., M.C., H.S., N.D., A.B. and A.C.M. Analysis and interpretation of data: A.S.F., J.E.R., M.W.R., S.D., M.F.D. and M.R. Supervision: R.F.P., P.B., A.P., M.W.R., S.D., M.F.D. and M.R. Writing (original draft): A.S.F., J.E.R. and M.R. Writing (review and editing): H.A., R.F.P., P.B., A.P., M.W.R., S.D. and M.F.D.
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A.S.F., J.E.R. and M.R. are inventors on a related patent application (PCT/US2024/020183) concerning the design and use of CD45-PET probes for detecting inflammation in inflammatory diseases. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 CD45-PET probe is highly specific to CD45.
a,b, Representative PET/CT (left) and PET (right) images of (a) healthy C57BL/6 mice (n = 4) imaged with the CD45-PET probe per standard protocol versus (b) healthy C57BL/6 mice (n = 4) that were pre-blocked with non-radiolabelled (“cold”) CD45 nanobody prior to probe administration. Spleen (asterisk), femoral bone marrow (arrow), and kidney (arrowhead) are denoted. See Supplementary Videos 4–6. c, Blocking results in significantly lower signal in immune cell-rich organs (Welch’s t-test: spleen p < 0.0001, bone marrow p = 0.0352, lymph node p = 0.0277, liver p = 0.0151) but no change in kidney signal (Welch’s t-test: p = 0.3792). See Supplementary Table 2 for absolute SUVmean values. d, SDS-PAGE analyses of irrelevant VHH4 nanobody, which targets human HLA Class II molecules and is thus non-targeting in mice. Lanes #1, protein marker with molecular weights (kDa) shown on the left; #2, VHH4 nanobody; #3, DFO-azide labelled VHH4 nanobody PEGylated with PEG20. e, Representative PET/CT (left) and PET (right) image of healthy C57BL/6 mice (n = 4) imaged with the 89Zr-labelled VHH4-DFO-PEG20 probe. Spleen (asterisk) and bone marrow signal (arrow) are not visualized, while non-specific kidney uptake (arrowhead) is observed. f, There is minimal non-specific probe uptake in immune-rich organs (Welch’s t-test: spleen p < 0.0001, bone marrow p = 0.0015, lymph nodes p = 0.0110) and organs with large resident immune cell populations (liver p = 0.0001, lung p = 0.0056) but not organs with few physiologic resident immune cells (heart p = 0.1145, colon p = 0.9422, muscle p = 0.0643). Excretion through kidneys produces similar signal between the two nanobody-based probes (p = 0.9423). See Supplementary Videos 7 and 8. Absolute SUVmean values available in Supplementary Table 3. All experiments performed once. All images are maximum intensity projections (MIPs). The PET and CT scale bars are in µCi mm−3 and Hounsfield units, respectively. All t-tests are two-tailed. In the bar graphs, the bars show the mean SUVmean and the error bars indicate the s.d.
Extended Data Fig. 2 Ex vivo studies validate CD45-PET inflammation detection in ARDS.
a, Lung biodistribution (%ID/g) 32 h after mice received intranasal LPS (n = 4 high-dose, n = 3 low-dose), with healthy mice as controls (n = 4). Lungs of high-dose mice emit significantly higher amounts of gamma radiation (a proxy for probe uptake) than the low-dose and control group (one-way analysis of variance with Tukey’s correction; p = 0.0001 and <0.0001, respectively). Two independent biological replicates performed. b, Phosphor screen images of CD45-PET probe in lungs from high-dose LPS (left) and control mice (right) show heterogeneous probe uptake within the LPS-exposed lung with regions of lower (Area 1) and higher (Area 2) uptake. Control lung uptake is uniformly low, represented by Area 3. c, Mouse CD45+ IHC performed on immediately adjacent sections, with corresponding 4X images for each area denoted in b. Areas of lower probe uptake shown (Area 1) has lower CD45+ staining compared to areas of higher probe uptake (Area 2), while both areas have higher CD45+ staining compared to control (Area 3). Insets are representative 40X images from each Area. Scale bars, 100 µm. Images represent 5 sections per mouse, experiment performed once. d,e, CD45+ IHC was performed on three non-adjacent sections of left lungs of high-dose (n = 4), low-dose (n = 3), and control mice (n = 4). The percentage of positively stained pixels per field of view strongly correlated with ex vivo lung SUVmean (d; r = 0.97, p = 0.0015) and biodistribution (e; r = 0.97, p = 0.0017). f, H&E lung injury scores for the high-dose LPS group was significantly higher than in low-dose LPS (p = 0.0002) and control mice (p < 0.0001), and in low-dose versus control mice (p = 0.0001) by one-way analysis of variance with Tukey’s correction. g,h, Representative H&E-stained lung samples from high-dose, low-dose, and control mice shown at 10X (g) and 40X (h). High- and low-dose LPS mice show substantial neutrophilic inflammatory infiltration. High-dose mice show infiltration of the alveoli (blue arrowhead) and interstitium (orange arrowhead), while inflammation in low-dose mice is largely confined to the interstitium. i,j, Correlation between probe uptake, weight loss (proxy of clinical severity) and lung injury scores of H&E samples (indicating histologic severity) was observed in both ex vivo lung SUVmean (i, weight loss: r = 0.94, p < 0.0001; lung injury score: r = 0.95, p < 0.0001) and biodistribution %ID/g (j, weight loss: r = 0.87, p = 0.0004; lung injury score: r = 0.83, p = 0.0015). Circle sizes represent the corresponding lung injury scores. In the bar graphs, the bars show the mean %ID/g or SUVmean and the error bars indicate the s.d. r, Pearson linear correlation coefficient.
Extended Data Fig. 3 CD45-PET detects bowel inflammation in IBD model and correlates with disease severity.
Acute colitis was induced in C57BL/6 mice with 4% DSS in drinking water for 7 days (n = 8, purple) or 5 days (n = 7, teal), with regular water controls (n = 4) in two independent biological replicates. Experimental setup is shown in Fig. 3a. a, At experimental endpoints, DSS-treated mice had significantly increased weight loss compared to controls. On day 5, mean weight change in DSS-treated mice was −9.67% ± 4.91% versus 0.85% ± 0.58% in controls (Welch’s t-test: p = 0.0011). On day 7, mean weight change in DSS-treated mice was −12.16% ± 2.47% versus 0.07% ± 0.61% in controls (Welch’s t-test: p < 0.0001). b, On the day of imaging, disease activity index (DAI) scores were 6.1 ± 2.7 (n = 7) after 5 days of DSS exposure and 7.7 ± 2.7 (n = 8) after 7 days of DSS exposure. c,d, Quantitative analysis of ex vivo PET images (c) and ex vivo biodistribution (d) of the 7-day DSS group confirms that DSS-exposed mice (n = 8) had higher large bowel probe uptake versus controls (n = 4) (Welch’s t-test: p = 0.0009 and p = 0.0034, respectively). e,f, Quantitative analysis of ex vivo PET images (e) and ex vivo biodistribution (f) confirm that large bowel probe uptake significantly correlates with percent weight loss from baseline (R = 0.80, p = 0.0004; R = 0.92, p = 0.0011, respectively). g,h, Quantitative analysis of in vivo PET images shows that while higher in vivo PET signal in the rectum was also correlated with weight loss (g; R = 0.61, p = 0.0178), it was not correlated with disease activity score (h; R = 0.31, p = 0.2521). All t-tests are two-tailed. In the bar graphs, the bars show the mean %ID/g or SUVmean and the error bars indicate the s.d. R, Spearman’s linear correlation coefficient.
Extended Data Fig. 4 CD45-PET captures response to treatment with water recovery with and without dexamethasone.
a–c, C57BL/6 mice (n = 14) were exposed to 2% DSS for 8 days before undergoing their first timepoint of CD45-PET imaging, with healthy C57BL/6 regular water controls (n = 4), representing one biological replicate. a,b, In vivo CD45-PET signal was significantly higher in both the large bowel (a, SUVmean 3.19 ± 1.51 vs 0.79 ± 0.33; Welch’s t-test: p < 0.0001) and rectum (b, SUVmean 4.61 ± 1.87 vs 0.69 ± 0.26; Welch’s t-test: p < 0.0001). c, No correlation was observed between rectum SUVmean and weight loss (r = 0.09). d–g, After completing the first imaging timepoint, mice were randomized to water recovery alone (n = 6) or water recovery with dexamethasone (n = 8, 1 mg/kg/day intraperitoneally for 3 days). One mouse in the water recovery alone group died before the second PT/CT imaging. CD45-PET imaging was repeated after 8 days of recovery on day 16. d,e, In vivo PET signal decreased across both groups between day 8 and day 16 in the large bowel (d, SUVmean 3.28 ± 1.53 vs. 1.40 ± 0.32; unpaired t-test: p = 0.0002) and rectum (e, SUVmean 4.81 ± 1.77 versus 2.27 ± 0.55; unpaired t-test: p = 0.0002). f,g, Percent change in SUVmean was not significantly different between water with dexamethasone and water-alone groups in the large bowel (f, SUVmean −51.9% ± 21.9% versus −47.6% ± 14.9%, respectively; Welch’s t-test: p = 0.7102) or rectum (g, SUVmean −43.2% ± 25.7% versus −54.2% ± 8.7%, respectively; Welch’s t-test: p = 0.3838). h, Weight change from day 8 to day 16 did not differ between mice on water and dexamethasone and water-alone regimens (mean weight gain 1.12% ± 3.84% versus 2.00% ± 1.87%, respectively; Welch’s t-test: p = 0.6469). i, The percentage of rectum SUVmean change is not correlated with the degree of disease recovery, as measured by the percentage of weight gain in regular water-only treated mice (n = 5) and regular water with dexamethasone-treated mice (n = 8) (r = −0.34, p = 0.2183). All t-tests are two-tailed. In the bar graphs, the bars show the mean %ID/g or SUVmean and the error bars indicate the s.d. r = Pearson’s linear correlation coefficient.
Extended Data Fig. 5 Whole body visualization of human immune cell infiltration using human CD45-PET.
NSG-MHCI/II dKO mice (n = 5) were injected with 10 × 106 human peripheral blood mononuclear cells (PBMCs) and underwent CD45-PET 21 days after humanization. Their development of graft-versus-host disease (GVHD) was monitored and they were imaged a second time to visualize human immune infiltration at day 112 post humanization. Data represent one biological replicate. a–d, Representative examples of unique imaging changes in individual GVHD mice across both timepoints. a, Day 21 imaging of Mouse 2 (left-sided panels) and Mouse 4 (right-sided panels). Clear signal can be seen in the spleen (asterisk). See Supplementary Videos 36 and 38. b, Four (M1-M4) of five mice developed one or more symptoms of GVHD by day 112, at which point CD45-PET imaging was repeated. c, Repeat day 112 imaging show decreased spleen signal (asterisk) and increased snout (Sn), kidney (Ki) and perineum (Per) signal across both Mouse 2 and Mouse 4 (teal arrows). However, not all organs showed uniform changes. Using colon and lung as an example in this figure, Mouse 2 showed increased large bowel signal localizing to the proximal colon (Mouse 2, yellow wedge), while Mouse 4 did not (Mouse 4, yellow wedge). In contrast, Mouse 4 showed subtle increases in lung signal (Mouse 4, purple wedge) whereas Mouse 2 did not (Mouse 2, purple wedge). See Supplementary Videos 37 and 39. d, Ex vivo images validate in vivo findings, where Mouse 4 lungs have higher signal than Mouse 2, with the reverse observed for colon. Further, signal differences can be clearly appreciated in heart (M2 > M4) and abdominal skin (M4 > M2). e, All mice that developed one or more GVHD symptoms (M1-M4) had a significant decrease in spleen signal between day 21 and 112 after humanization, while the mouse without symptoms did not (F1). f,g, Change in vivo PET signal in the colons (f) and lungs (g) of each mouse (M1-M4, F1) between day 21 and 112. To determine whether signal increases reflected non-specific signal secondary to spleen loss or true graft infiltration, the upper limit of the 95th percent confidence interval (95% CI) of the control signal for each organ was applied to each as a positive signal threshold (orange dotted line). Additional organs analyzed shown in Supplementary Fig. 10. h,i, Human CD45 IHC staining was performed on the proximal colons (h, 20X) and right lungs (i, 10X) of all mice (n = 3 non-adjacent sections per organ). As predicted by in vivo PET analyses shown in f,g, Mouse 2 had human CD45+ cells in the proximal colon localizing to areas with above-threshold signal (h, top panel inset red arrows). Mouse 2 had no immune infiltration in the lung (i, top panel). Similarly, Mouse 4 showed below-threshold signal in the proximal colon and had no immune infiltration (h, bottom panel). However, above-threshold signal in Mouse 4’s lung corresponded to increased human CD45+ cells and gross changes in lung morphology (i, bottom panel inset red arrows). Scale bars 200 µm (h), 30 µm (h inset), 400 µm (i), and 40 µm (i inset). All images are maximum intensity projections (MIPs). D21, Day 21; D112, Day 112; Sn, snout skin; Ki, kidney; Per, perineum; Lu, lung; R Lu, right lung; L Lu, left lung; H, heart; M, muscle; Co, colon; Ab, abdominal skin; Δ, change.
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Salehi Farid, A., Rowley, J.E., Allen, H.H. et al. CD45-PET is a robust, non-invasive tool for imaging inflammation. Nature (2025). https://doi.org/10.1038/s41586-024-08441-6
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DOI: https://doi.org/10.1038/s41586-024-08441-6
