Non-invasive imaging with ICOS-targeting monoclonal antibody for preclinical diagnosis of rheumatoid arthritis in a humanized mouse model

doi:10.21203/rs.3.rs-5080168/v1

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Research Article

Non-invasive imaging with ICOS-targeting monoclonal antibody for preclinical diagnosis of rheumatoid arthritis in a humanized mouse model

https://doi.org/10.21203/rs.3.rs-5080168/v1

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Background

Activated T cells play a pivotal role in rheumatoid arthritis (RA) pathogenesis, and imaging of activated T cells may provide a non-invasive tool for RA detection. Here, we first developed an optical probe targeting human ICOS and tested its capacity in RA diagnosis by capturing ICOS⁺ activated T cells in vivo in a humanized mouse model.

Methods

The humanized arthritis model, HuPBMC-AIA (Human peripheral blood mononuclear cells- adjuvant induced arthritis) was established, and flow cytometry and immunofluorescence were employed to determine ICOS expression in huPBMC-AIA model. Anti-human ICOS monoclonal antibody (mAb) was conjugated to Cy7 via NHS ester amine reaction. A cell uptake study was used to confirm the specificity of Cy7-ICOS mAb to activated T cells. 4-view NIRF imaging study was performed to test Cy7-ICOS mAb in detecting RA in vivo.

Findings

ICOS was confirmed as an indicator of RA pathogenesis via RNA-seq, flow cytometry and immunofluorescence data. An in-vitro cellular uptake study validated the specificity of Cy7-ICOS mAb to activated T cells. Cy7-ICOS mAb could detect ICOS⁺ activated T cells in vivo through 4-view NIRF imaging. The receiver operating characteristic (ROC) curve created based on NIRF imaging quantification could distinguish the huPBMC-AIA group from the control group at all time points imaged.

Conclusion

In this study, we first developed an optical imaging probe targeting human ICOS, Cy7-ICOS mAb. The 4-view NIRF imaging with Cy7-ICOS mAb could detect pathogenic ICOS⁺ activated T cells with high sensitivity and specificity in vivo, which indicated the great potential of this imaging probe in RA early diagnosis.

ICOS

NIRF imaging

rheumatoid arthritis

humanized model

activated T cell

the first to target human ICOS, a critical aspect in advancing towards humanized immunology.

ICOS as a promising biomarker for early RA detection.

the validity of the huPBMC-AIA model in mimicking the complex microenvironment of RA.

Our findings offer a paradigm shift in the approach to RA research, suggesting that humanized models, particularly those targeting ICOS, provide a more clinically relevant platform for early diagnosis. The simplicity of utilizing a single biomarker for imaging in the complex microenvironment of RA holds promise for facilitating easier and more effective diagnosis, thus potentially improving RA patient management.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by debilitating, chronic, and debilitating joint inflammation [1]. Early diagnosis and timely intervention may lead to better prognosis of rheumatoid arthritis patients [2]. Due to the low sensitivity and specificity, current approaches often fail in RA early detection [3]. For instance, anatomic imaging techniques, can only diagnose RA when cortical bone destruction has occurred. On the other hand, blood tests of RA-specific autoantibodies, such as rheumatoid factor (RF) and anticitrullinated protein antibodies (ACPA), often lack specificity [4]. To overcome these challenges, there is an urgent need to develop novel tools for RA early detection.

Autoreactive T cell activation gone awry has been proposed as the leading cause of autoimmune disorders, such as multiple sclerosis (MS) and rheumatoid arthritis (RA). During RA pathogenesis, CD4⁺ T cells stimulate osteoclast and macrophage activation, autoantibody synthesis, lymphoid neogenesis, and pannus development in the inflammatory joint [5]. It is thought that activation of major histocompatibility complex (MHC) class II-restricted autoreactive T cells, which specifically assist B cells in producing autoantibodies, is the cause of RA years before the disease manifests clinically [6]. Moreover, T-cell activation is enriched in synovial tissue, which predates the onset of RA [7]. As consequently, techniques to precisely identify and monitor activated T cell reactions in vivo may assist with RA diagnosis, staging, and treatment monitoring. Molecular imaging has shown great potential for achieving this goal [8]. A nucleoside analogue, 2′-deoxy-2′-[18F]fluoro-9-β-D-arabinofuranosylguanine ([18F]F-AraG), was tested in RA detection in a murine AIA-induced arthritis model [9], while despite the high uptake of activated T cells, [18F]F-AraG also accumulated in other immune cells, such as macrophages and dendritic cells. This unexpected result may be attributed to the same nucleic acid metabolic pathways shared by immune cells [10].

Thus, we diverted our attention to T-cell surface costimulatory receptors. In our previous study, we developed an NIRF imaging probe targeting murine 4-1BB, and we successfully diagnosed RA pathogenesis at early stages via in vivo activated T cell tracking [11], which demonstrated the feasibility of imaging T-cell surface costimulatory receptors in RA early diagnosis. In consideration of further clinical translation, as well as to select the most sensitive biomarker for RA imaging, we analyzed RNA-seq data from the SRA database, and inducible T-cell costimulatory receptor (ICOS or CD278) was chosen for this study [12]. ICOS is a member of the CD28 superfamily, which is mainly expressed on activated T cells. The ligation of ICOS and its ligand (ICOSL) could activate the downstream pathway and regulate T-cell proliferation, as well as cytokine secretion[13]. It had been proved as an ideal biomarker for in vivo activated T cell tracking in many murine models [14–16], whereas no humanized model or clinical studies were reported. Here, we first assessed human ICOS imaging in a humanized rheumatoid arthritis model. ICOS expression was validated via flow cytometry and immunofluorescence staining. An optical imaging probe targeting human ICOS, Cy7-ICOS mAb was synthesized and evaluated via both in vitro cell uptake and in vivo NIRF imaging studies. Our findings demonstrated that ICOS targeted NIRF imaging is a promising strategy for rheumatoid arthritis early diagnosis.

Data Source

Raw RNA-seq data for GEO dataset GSE89408 was downloaded from the SRA database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE89408) and quantified to gene-level counts using the ARCHS4 pipeline [17]. GSE89408 dataset, which included 57 early RA samples and 28 normal samples, were selected as the validation sets. Gene counts were downloaded from the ARCHS4 gene expression matrix v5. For more information about ARCHS4, as well as free access to the quantified gene expression matrix, visit the project home page at the following URL: http://amp.pharm.mssm.edu/archs4/download.html.

Data Normalization

Raw counts were normalized to log10-Counts Per Million (logCPM) by dividing each column by the total sum of its counts, multiplying it by 10⁶, followed by the application of a log10-transform.

Volcano plot analysis

Volcano plot was plotted by https://www.bioinformatics.com.cn (last accessed on 10 July 2023), an online platform for data analysis and visualization.

Immune infiltration analysis

The deconvolution method was employed to process marker gene expression values to estimate the proportion of different types of immune cells in the glomerular tissues of both normal and diabetic nephropathy. The CIBERSORT algorithm was used for analysis. These cells comprise 22 different types of immune cells, including M1 and M2 macrophages, plasma cells, static memory CD4 + T cells, γδT cells, and mast cells. For every sample, a boxplot representing the percentage of T cell was created.

Cell culture and huPBMC-AIA model

Human peripheral blood mononuclear cells (HuPBMC) from Charles River (formerly HemaCare). huPBMC was kept at 37°C in a humidified incubator with 5% CO₂. Harvested during the exponential growth phase, the cells were resuspended at a density of 10x10⁶ cells/mL in full growth media.

All the animal studies were conducted under protocols approved by the Local Ethical Committee of Harbin Medical University Animal Care and Use. Five-week-old NOG male (Charles River, USA) mice were infused intravenously with 3x10⁶ huPBMC. Peripheral blood was collected, and flow cytometry was used to evaluate the immune cell subpopulations to verify the humanization. The humanized mice were included in the Adjuvant induced arthritis (AIA) induction protocol after achieving sufficient immune blood reconstitution (engraftment efficiency (huCD45 > 25%)).

For the humanized arthritis model, the periarticular space surrounding the ankle was injected with 10 µL of either complete Freund's adjuvant (CFA) 4 mg/mL (Chondrex, Redmond, WA) or PBS. To deplete murine neutrophils, anti-Gr-1 antibody (250µg) (BioXCell, West Lebanon, US) was given intraperitoneally (IP) on days 1 and − 1. Ankle joint processing and arthritis were carried out as previously mentioned [18]. A vernier caliper was used to measure the thickness of the paw.

RNA-seq Analysis and KEGG

Total RNA was extracted from paw of huPBMC-AIA model followed by library preparation according to Illumina standard instruction (VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina®). Agilent 4200 bioanalyzer was employed to evaluate the concentration and size distribution of cDNA library before sequencing with an Illumina novaseq6000. The protocol of high-throughput sequencing was fully according to the manufacturer's instructions (Illumina). The raw reads were filtered by Seqtk before mapping to genome using Hisat2 (version:2.0.4). The fragments of genes were counted using stringtie(v1.3.3b) followed by TMM (trimmed mean of M values) normalization. Significant differential expressed genes (DEGs) were identified as those with a False Discovery Rate (FDR) value above the threshold (Q < 0.05) and fold-change > 2 using edgeR software [19]. An online platform (chiplot) was used for the KEGG pathway and Reactome enrichment analyses of DEGs.

Flow Cytometry

Flow cytometric analyses were performed on a flow cytometer CytoFLEX (Beckman Coulter, USA). The peripheral blood, spleen, bone marrow, and paw were processed in the same manner as previously described [18]. Single cell suspensions were stained with a mixture of fluorochrome-conjugated antibodies against human CD45, human CD4, human CD8, human CD278, mouse CD11b, mouse Gr-1, and mouse CD45, after being fixed with the Zombie AquaTM Fixable Viability Kit (BioLegend, San Diego, CA). FlowJo software (Treestar Inc, OR) and online platform (Cytobank, Beckmen, US) was used for the analyses.

Conjugation of Cy7-ICOS mAb

The supplier of the in vivo monoclonal antibody (clone: C398.4A) against human ICOS was BioXCell, located in West Lebanon, USA. The Cy7 NHS ester was supplied by APExBIO. Anti-ICOS mAb was diluted in PBS to 1 mg/mL. Cy7 NHS ester was dissolved in dimethyl sulfoxide to a final concentration of 5 mg/mL before being mixed with anti-ICOS mAb. The ICOS mAb to Cy7 NHS ester molar ratio was 1:10. The mixture was incubated at room temperature for 1 hour. Cy7-ICOS mAb was purified using a Vivaspin2 50 KDa cutoff MWCO spin filter (GE Healthcare, Piscataway, USA). NanoDrop (Thermo Fisher, Waltham, MA, USA) was used to determine the final concentration of Cy7-ICOS mAb.

Cell uptake study

Dulbecco's medium (IMDM). The 500X concentrate of the Cell stimulation Cocktail (Invitrogen, Carlsbad, USA) was added straight to the culture medium at a rate of 1X (2 uL/mL) to activate T cells. Following 1-hour incubation at 37°C, 2µg of Cy7-ICOS mAb was added after 48 hours of stimulation. Before the binding assay, activated T cells were treated with 100 µg of unconjugated ICOS mAb and incubated for 1 hour at 37°C for the blocking group. A multiscan spectrum (Tecan, Switzerland) and a CytoFLEX flow cytometer were used to analyze the data.

In vivo 4-view Near-infrared fluorescence imaging (NIRF) studies

An OPTICAL IMAGING IN VISIBLE & NIR I (Biospace, California, USA) was applied to collect the NIRF imaging data. On the 3rd day following the induction of arthritis, 50 µg of Cy7-ICOS mAb was administered via the mouse tail vein following anesthesia with 2–3% isoflurane gas. On days 3, 4, 5, 6, and 7, NIRF imaging was performed using the following parameters: Acquisition mode: FLI integration (1000 ms per frame − 5 frames) Fluorophore: Cy7, Excitation = 737 nm, Background = 687 nm, Emission = 797 nm, Filter cut off = 780–815 nm (band pass) nm; Lens: 50mm f/1.2; Aperture = 1.2; Focus = 93.0. Overlaying x-ray images and optical signals is possible with the PhotonIMAGER Optima's X-ray module. Improved optical signal localization and quantification are provided by the 4-View – 3D module (Acquisition mode: FLI integration (1000 ms per frame − 5 frames Fluorophore: Cy7, Excitation = 737 nm, Background = 687 nm, Emission = 797 nm, Filter cut off = 780–815 nm (band pass) nm Lens: 35mm f/1.4; Aperture = 1.4; Focus = 94.0). Region of interest (ROI) of NIRF images was drawn using software (M3 vision, Biospace, California, USA).

Ex Vivo Biodistribution

To validate the ROI data, mice were euthanized after the imaging session on day 7 and subjected to the standard ex vivo biodistribution analysis described above. The following organs were harvested: the heart, liver, lung, spleen, kidney, intestine, right paw (RP), left paw (LP), bone, muscle, skin, and blood. Parameters for acquisition: 50mm f/1.2 lens; 1.2 aperture; 96.0 focus.

Immunofluorescence

Immunofluorescence (IF) staining antibodies: Abcam (Boston, USA) provided the IgG H&L (Alexa Fluor® 488) (ab173003) and IgG H&L (Alexa Fluor® 594) (ab150108). BioLegend provided a purified anti-human CD3 antibody (Clone: OKT3) and an anti-human/mouse/rat CD278 (ICOS) antibody (Clone: C398.4A). After the mice were sacrificed, the ankle joints were fixed with 4% paraformaldehyde and decalcified for 40 days with 10% EDTA. After embedding in paraffin After deparaffinization, antigen retrieval, and BSA blockade, ankle joint sections were stained overnight at 4°C with CD3 or CD278 antibodies. The fluorescent-labeled secondary antibodies were then added and incubated for 1 hour. After staining the nuclei with DAPI (Beyotime, Shanghai, China), the sections were examined under a fluorescent microscope (Nikon, Japan).

Safety Evaluation

During the procedures, the body weight was recorded. Blood was collected three days after the last administration, and serum was separated by centrifugation. Standard kits were used to measure serum levels of ALT, AST, BUN, and Crea. Major organs (heart, liver, spleen, lung, and kidney) were removed, fixed with 4% paraformaldehyde, and stained with hematoxylin and eosin (H&E). A Light microscope (Nikon, Japan) was then used to image the sections.

Statistical Analysis

An online platform for data analysis and visualization, https://www.chiplot.online, was used for data management and analysis. For the statistical analyses, GraphPad Prism 9.0v (GraphPad, CA, USA) software was utilized. Our tests have not been parametric. There were only two groups compared using the Mann-Whitney test. P-values below 0.05 were regarded as statistically significant.

T cell portion profile and differential expression analysis (DEG) in early rheumatoid arthritis

To select the most sensitive imaging biomarker for RA, the GSE89408 dataset was chosen for T cell portion analysis and DEG identification. It contained 57 early RA and 28 healthy control (HC) samples from synovial tissues. We employed the CIBERSORT to study immune infiltration in this dataset. Compared to HC group, activated CD4⁺ memory T cells were found to be increased in early RA group, along with decreased CD4⁺ memory resting T cells, P < 0.01 (Fig. 1A), which indicated that activated T cells are driven factors of rheumatoid arthritis at an early stage [20]. Further DEG data was visualized using volcano plot analyses (Fig. 1B), ICOS was the most differential activated T cell genes among all 275 DEGs in RA samples, compared to other T cell genes (TNFRSF 9, TNFRSF 4, CD69 and CTLA 4). To validate the expression of ICOS on activated human T cells, human PBMCs were incubated with cell-stimulating kit (PMA/IONO) for 72 hours. According to the flow cytometry study, a significant peak shift could be observed in PMA/IONO group (Fig. 1C). Higher frequency of ICOS could be detected on both CD4 and CD8 T cells, compared to PMA/Iono + blocked, blocked, and resting groups, P < 0.001 (Fig. 1D and 1E).

Establishment and Validation of huPBMC-AIA rheumatoid arthritis model

The animal study scheme is shown in Fig. 2A. Generally, Human PBMC was injected into NOG mice via the tail vein to establish the huPBMC-AIA model. Weekly peripheral blood was taken to confirm humanization using flow cytometry. According to our data (Freq.huCD45(%) -2week: 0.68 ± 1.66; -1 week: 10.34 ± 5.55; day0: 35.61 ± 13.26), the engraftment efficiency reached to a high level after three weeks post PBMC injection (Fig. 2B and 2C), which validated the success of NOG mice humanization. The depletion of Gr-1 was carried out at day 1 and − 1 to minimize the intervention of myeloid cells on inflammation [20]. The CD11b⁺Gr-1⁺ cell frequency from in vivo depletion of Gr-1⁺ group was much lower than that in the control group, P = 0.008 (Fig. 2D). Following CFA injection, the symptoms, such as swelling and redness, could be observed in inflamed joint from huPBMC-AIA group (Fig. 2E). To monitor the severity of joint arthritis, paw thickness was measured every day after CFA injection, and the paw thickness in the huPBMC-AIA model was higher than that in the control group at all time points examined (P < 0.01) (Fig. 2F). To determine whether huPBMC-AIA model could accurately mimic RA pathogenesis, RNA-seq was performed using CFA injected paw. To reveal all the changes (especially immunology-related reactions) and define KEGG disease, we performed transcriptome sequencing on treated paws 3 days after adjuvant injection. huPBMC-AIA mode could promote the expression of specific genes related to RA. (Fig. 2G).

ICOS is an indicator of RA pathogenesis at early stage in huPBMC-AIA model

IF staining was conducted on the day3 to determine ICOS expression in injected paw of both control and huPBMC-AIA model. Confocal imaging confirmed positive expression of both CD3 and ICOS in huPBMC-AIA group, whereas no obvious signal could be detected in the control group. Good co-localization of CD3 and ICOS was also observed in huPBMC-AIA group (Fig. 3A). To re-confirm the consistent role of ICOS during RA pathogenesis, flow cytometry study was performed at day7. The most striking finding beyond our thought was that ICOS expression in bone marrow (BM) was much higher in huPBMC-AIA group (huCD45⁺CD4⁺ICOS⁺|Freq.of Parent: P < 0.01; huCD45⁺CD8⁺ICOS⁺|Freq.of Parent: P < 0.05) (Fig. 3B), which indicated CFA injection may also lead to systemic immune response. ICOS⁺CD4⁺ frequency in CFA injected paw from huPBMC-AIA group was considerably higher compared to control group, which was in line with IF staining data. tSNE plot demonstrated ICOS is strict to T cells (Fig. 3C). As an indicator of RA pathogenesis, there was a significant positive correlation between paw thickness and huCD45⁺CD4⁺ICOS⁺ T cells (P value = 0.0001; R square = 0.8548) (Fig. 3D). Principal component/components analysis (PCA) significantly separated animals from the huPBMC-AIA group from other mice throughout PC1, which accounted for 52.4% of the variance (Fig. 3E). The major contributors in identifying mice in the huPBMC-AIA group from other cohorts over PC1 were paw CD4⁺ICOS⁺, BM CD4⁺ICOS⁺, and BM CD8⁺ICOS⁺. These findings suggested that the invasion of ICOS⁺ activated T cells was primarily responsible for the early modeling of huPBMC-AIA.

Conjugation and Characterizations of Cy7-ICOS mAb

The anti-human ICOS mAb was conjugated with Cy7-NHS ester, then purified using a Vivaspin2 50 KDa cut-off spin filter (Fig. 4A). The multiscan spectrum was used to test the probe absorbance (Fig. 4B). Based on the results of three separate trials, the chemical yield of the probe was 65%. To assess the binding of Cy7-ICOS mAb to activated T cells, in vitro cell uptake assays were conducted. There was no obvious difference at 1nM and 10nM of Cy7-ICOS mAb concentration, whereas significant difference could be detected at 100nM level (p < 0.01). In contrast to blocked or naïve cohorts, the ICOS MFI was much higher in activated T cell group (p < 0.001) (Fig. 4C and 4D).

Cy7-ICOS mAb detected activated T cells in huPBMC-AIA model via NIRF imaging

The 4-view NIRF imaging design and ROI atlas were shown in Fig. 5A. On day3, each mouse was received 50 µg Cy7-ICOS mAb administration via the tail vein, and longitudinal NIRF/X-ray imaging was carried out 6, 24, 48, 72, and 96 hours post injection. 4-View images and Top NIRF/X-ray images were listed in Fig. 5B and 5C. As we expected, significant optical signals could be observed at injected right paw (RP) of hu-PBMC AIA group at all time points examined. The Cy7-ICOS mAb pharmacokinetics at RP from 4-view images are shown in Fig. 5D. From 6 hours to 96 hours, the RP fluorescence intensity of huPBMC-AIA was consistently higher than that in control group (6h: p < 0.01; 24h: p < 0.01; 48h: p < 0.01; 72h: p < 0.001; 96h: p < 0.0001). The ROI profiles of the huPBMC-AIA and control groups were compared using fluorescence intensity ratio of RP to LP. The huPBMC-AIA group RP/LP ratio was noticeably higher than that in control group (huPBMC-AIA: 6h = 1.62 ± 0.23; 24h = 1.59 ± 0.23; 48h = 1.43 ± 0.19; 72h = 1.27 ± 0.09; 96h = 1.25 ± 0.08; control: 6h = 1.04 ± 0.04; 24h = 0.87 ± 0.06; 48h = 1.08 ± 0.11; 72h = 1.06 ± 0.09; 96h = 1.06 ± 0.10 and P value = 0.000171, 0.000024, 0.002716, 0.006593, respectively) (Fig. 5E). A strong correlation between paw thickness and RP/LP ratio could be detected with all time points ROI data (P value < 0.0001; R squared = 0.71) (Fig. 5F). As an optical imaging probe based on monoclonal antibody, Cy7-ICOS mAb should not exacerbate RA pathogenesis. To assess Cy7-ICOS mAb safety, the same dose of control mAb was also administrated. While there was no difference of paw thickness and body weight observed between two cohorts (Supplementary Fig. 1A and 1B). Blood urea nitrogen (BUN), creatinine (Crea), aspartate aminotransferase (AST), and alanine transaminase (ALT) were also measured as representative blood biochemical indicators (Supplementary Fig. 1C and 1D). The major organs were further stained for histological analysis (Supplementary Fig. 1E), and no pathological change was observed.

Evaluation of ICOS imaging metrics for RA diagnosis at early stage

Ex vivo NIRF imaging was performed immediately after the final imaging at day 7 to validate the accuracy of our manual ROI measurement (Fig. 6A). Among all the organs collected, the RP FI in huPBMC-AIA group was higher than control group (P < 0.05), which is consistent with in vivo NIRF imaging data (Fig. 6B). Intra-group correlation and cluster analysis of in vitro distribution data showed that RP was clustered significantly with liver, while RP was negatively correlated with kidney (p < 0.05) (Fig. 6C). This indicates that ICOS-mAb exhibits selectivity in the paw caused by arthritis and complies with the hepatic absorption metabolism. The NIRF and BioD data showed good concordance at all RP locations (Pearson correlation: 6h = 0.91; 24h = 0.88; 96h = 0.67) (Fig. 6D). PCA showed that PC1 contributed 92.95% and PC2 contributed 4.04%, which indicated that the NIRF signal from RP was the major mediator to distinguish RA from control cohort. (Fig. 6E). ROI values from dimension reduction analysis at various time stages demonstrated that the preclinical imaging of the control and huPBMC-AIA groups could be clearly identified. The huPBMC-AIA could be distinguished from control group using receiver operating characteristic (ROC) curves based on FI RP/LP ratio at all time points photographed (P < 0.0001; area under the curve (AUC) = 0.9778) (Fig. 6F), which demonstrated the capacity of ICOS NIRF imaging in RA early diagnosis.

The life quality of rheumatoid arthritis patients could be improved via early diagnosis and intervention [21]. Given the low specificity and sensitivity, current approaches often fail in RA early detection. Thus, the development of novel diagnostic tools is essential. In this study, we identified ICOS as an indicator of RA pathogenesis, and NIRF imaging targeting ICOS enables precise detection of RA at early stages.

For selecting the most sensitive and specific cell portion and biomarkers for RA imaging, we first employed RNA-seq omics data. Activated T cells were identified as the primary cell portion in early RA samples, which was in line with our knowledge. Within the same dataset, ICOS was picked up as one of the most differential genes among all 275 DEGs identified, and moreover, ICOS outperformed other T cell costimulatory receptors, such as TNFRSF 9 (4-1BB) and TNFRSF 4 (OX40), this will help reduce the arguments about selecting biomarkers for activated T cell tracking. In addition, previous studies noted that abnormal ICOS expression was correlated with the pathogenesis of RA [22]. Further flow cytometry and immunofluorescence staining data from protein level matched with the omics data from genetic level. A strong correlation between paw thickness and ICOS expression level strengthened the hypothesis that ICOS was an ideal biomarker for RA diagnosis and severity monitoring. Based on the data listed, the NIRF imaging data acquired should be attributed to the massive infiltrated activated T cells in the ankle joint from huPBMC-AIA model, which makes ICOS imaging with high specificity. The NIRF imaging pattern we chose for this study is widely applied in surgery guiding and superficial disease imaging [23], and other advantages, such as non-radioactive, safe and low cost, enables NIRF imaging more suitable for clinical translation. Through NIRF imaging, we could visualize the dynamics of activated T cells in a non-invasive manner. With ROI quantification, we could easily distinguish RA mice from control cohort at all time-point imaged, which demonstrated NIRF imaging targeting ICOS was a reliable tool for RA detection. Ex vivo Pearson, PCA and ROC analysis further reinforced the thesis. Thus, with this non-invasive imaging technique, physicians could optimize RA patient management in clinic.

Fundamental distinctions of immune systems between mice and human hamper the translation of basic research results to the bedside [24]. Multiple editorials and assessments on the present status of immunology research have recently emphasized the demand for humanized immunology rather than murine immunology [25]. Previous T cell costimulatory receptors imaging studies were all targeting murine activated T cells, and to our knowledge, this is the first study of targeting human ICOS. The development of humanized systemic RA models will allow us to get greater promise for ICOS in RA identification at early stage, which is associated with its closer resemblance to the human immune systems. The NOG mice we chose in this study have a complement C5 component deficiency, which leads to diminished activation of the remnant mouse immune system [26]. Total RNA-seq study with CFA injected paw and KEGG analysis emphasizes the validity of huPBMC-AIA model. One of the most promising avenues for future research lies in extending the application of T cell imaging probes to other autoimmune conditions. The adaptability of our imaging approach suggests its potential utility in diseases such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis, among others. Investigating T cell behavior in diverse autoimmune contexts can uncover commonalities and unique aspects of immune responses, facilitating a more comprehensive understanding of autoimmunity.

ICOS imaging study presented here does have limitations. The flow cytometry data had indicated the increased expression of ICOS in bone marrow, while with NIR-I imaging dye Cy7, we failed to capture this signal in vivo. To further improve our work, we will employ NIR-II dye (1000–1700 nm) for deeper tissue imaging. Another limitation was that CFA injection could only induce acute inflammation, and the foot swelling appeared too fast. Thus, the hypothesis to acquire NIRF images before RA symptoms showing up could not be realized in this model. In the future, we should test collagen antibody-induced arthritis (CAIA) model, which may provide a chronic inflammation microenvironment.

In this proof-of-concept work, we successfully identified ICOS as a sensitive and specific biomarker for RA detection, and NIRF imaging with Cy7-ICOS mAb was a promising approach in early RA diagnosis. Among the complex microenvironment of RA, one single biomarker imaging could make the diagnosis easier and simple, which may provide a better choice for RA patients management. Our findings suggest ICOS imaging techniques are warrant further evaluation in the clinic.

The current study follows all applicable guidelines (international, national, and/or institutional) for the care and use of animals.

The authors would like to acknowledge the Shanghai Biotechnology Corporation for their assistance with the mRNA sequencing study.

Funding

This work was supported by the National Natural Science Foundation of China (82272056, 82472036), Heilongjiang Postdoctoral Scientific Research Developmental Fund (LBH-Q21136), Natural Science Foundation of Heilongjiang Province (LH2023H045), The Ningbo Major Research and Development Plan Project (20242214), Ningbo Yongjiang Talent Programme, Heilongiiang Medical Development Foundation and Research Project of Suzhou Gusu Health Talents Program (GSWS2022136).

Conflict of interest

The authors declare they have no financial interests.

Data availability

The authors declare that (the/all other) data supporting the findings of this study are available within article (and its supplementary information files)

Author contributions

Chao Li and Shao Duan conceived and designed research studies, developed methodology, conducted experiments, acquired data, analyzed data, and prepared the manuscript. Feng Yan, Yifei Xia and Shuaiming Shao conducted the experiments. Weiyu Chen and Zunyu Xiao conceived and designed the research studies and prepared the manuscript.

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Supplementary Figure 1 A and B Body weight and paw thickness was observed between ICOS mAb (n=6) and control mAb groups (n=6). C and D The blood biochemical indexes of Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST), as well as Blood Urea Nitrogen (BUN) and Creatinine (Crea), were systematically analyzed. E Hematoxylin and Eosin (H&E) staining were meticulously analyzed for major organs, including the heart, liver, spleen, lung, and kidney. Scale bar=100μm. All values represent the mean ±SEM unless otherwise specified. One-way ANOVA was conducted for statistical significance. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns p>0.05
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Editorial decision: Major revision
31 Oct, 2024
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17 Oct, 2024
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You are reading this latest preprint version

Non-invasive imaging with ICOS-targeting monoclonal antibody for preclinical diagnosis of rheumatoid arthritis in a humanized mouse model

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Version 1

Abstract

Figures

Highlights

Context and significance

Introduction

Material and methods

Data Source

Data Normalization

Volcano plot analysis

Immune infiltration analysis

Cell culture and huPBMC-AIA model

RNA-seq Analysis and KEGG

Flow Cytometry

Conjugation of Cy7-ICOS mAb

Cell uptake study

In vivo 4-view Near-infrared fluorescence imaging (NIRF) studies

Ex Vivo Biodistribution

Immunofluorescence

Safety Evaluation

Statistical Analysis

Results

T cell portion profile and differential expression analysis (DEG) in early rheumatoid arthritis

Establishment and Validation of huPBMC-AIA rheumatoid arthritis model

ICOS is an indicator of RA pathogenesis at early stage in huPBMC-AIA model

Conjugation and Characterizations of Cy7-ICOS mAb

Cy7-ICOS mAb detected activated T cells in huPBMC-AIA model via NIRF imaging

Evaluation of ICOS imaging metrics for RA diagnosis at early stage

Discussion

Declarations

References

Supplementary Files

Status:

Version 1