Lymph node macrophage-targeted interferon alpha boosts anticancer immune responses by regulating CD169-positive phenotype of macrophages

doi:10.21203/rs.3.rs-4946728/v1

Download PDF

Research Article

Lymph node macrophage-targeted interferon alpha boosts anticancer immune responses by regulating CD169-positive phenotype of macrophages

https://doi.org/10.21203/rs.3.rs-4946728/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

CD169⁺ macrophages in lymph nodes (LNs) activate cytotoxic T lymphocytes (CTLs), which play a crucial role in anticancer immunity, through antigen presentation and co-stimulation by CD169. Interferon alpha (IFNα) is capable of inducing the CD169⁺ phenotype of macrophages; however, its clinical applications have been hindered by pharmacokinetic limitations—low LN distribution and an inability to target macrophages. To overcome these issues, this study genetically fused mouse IFNα (mIFNα) with mannosylated mouse serum albumin (Man-MSA), and investigated the antitumor effects of the hybrid protein (Man-MSA-mIFNα) or its add-on effects with programmed death-ligand 1 (PD-L1) blockade.

Methods

To confirm CD169⁺ macrophage-mediated T cell priming, positional information about individual immune cells in LNs of cancer patients was obtained. Traits of Man-MSA-mIFNα, which was prepared using Pichia pastoris to form the high-mannose structure, were characterized by several physicochemical methods. To evaluate the lymphatic drainage of Man-MSA-mIFNα, radioiodine or Cy5-labeled Man-MSA-mIFNα was subcutaneously administered in mice, and then the radioactivity or fluorescence in LNs was analyzed. CD169-diphtheria toxin (DT) receptor (CD169-DTR) mice in which LN CD169⁺ macrophages can be depleted by DT injection were used to verify whether the antitumor effect of Man-MSA-mIFNα is dependent on LN CD169⁺ macrophages.

Results

Multiplex tissue imaging predicted close proximity of CD169⁺ macrophages and T cells and positive correlation between the number of CD169⁺ macrophages and T cells in neighborhoods in LNs of cancer patients. Physicochemical analyses showed that Man-MSA-mIFNα was formed from the fusion of the intact Man-MSA and mIFNα. Man-MSA-mIFNα efficiently induced the CD169⁺ phenotype of macrophages by its high LN distribution and macrophage-targeting capability, and significantly exerted antitumor activity through CTL activation in the LNs, whereas its antitumor effects were canceled in CD169-DTR mice. Finally, combination therapy with PD-L1 blockade markedly suppressed tumor growth in MB49-bearing mice, which exhibit resistance to PD-L1 blockade monotherapy.

Conclusions

The present study successfully designed and developed Man-MSA-mIFNα, which efficiently induces the CD169⁺ phenotype in LN macrophages, contributing to the antitumor immunity. The findings suggest that our novel strategy targeting CD169⁺ macrophages could be a promising immunotherapy for cancer patients who are unresponsive to immune checkpoint inhibitors.

lymph node

macrophage

CD169

albumin

IFNα

drug delivery system

cancer immunotherapy

targeted therapy

Tumor immunotherapy enhances the body's natural immune system to combat cancer. This type of treatment leverages innate immunity, including macrophages and dendritic cells, and acquired immunity, encompassing T and B cells. Activated cytotoxic T lymphocytes (CTLs) play a crucial role in cancer cell destruction by releasing inflammatory cytokines such as interferon-gamma (IFNγ), perforin, and granzymes. Studies show that an increased presence of CTLs in tumors is linked to improved patient outcomes [1]. Extensive research has been done on molecules and applications that stimulate CTLs. One established method involves immune checkpoint inhibitors that activate CTLs by targeting immune checkpoint molecules on the surfaces of cancer and immune cells, including macrophages. The use of antiprogrammed death-1/ligand-1 (anti-PD-1/PD-L1) antibodies is approved for treating various cancers, such as malignant melanoma, non-small cell lung cancer, and renal cell carcinoma [2]. Drugs targeting other immune checkpoint molecules are also available and have proven effective in cancer treatment. Ongoing research in this field aims to refine methods for CTL activation to enhance the efficacy of cancer immunotherapy.

Understanding the mechanisms underlying CTL activation is crucial for developing cancer immunotherapy methods. Mellman et al. synthesized their insights into the CTL induction process and developed the concept of the "cancer-immunity cycle [3]." According to this model, dendritic cells play a significant role in CTL activation. However, Asano et al. uncovered findings that diverge from this theory. Their research involved administering dead cancer cells to mice and monitoring their movement within the lymphatic system. They reported that CD169⁺ macrophages, not dendritic cells, were mainly responsible for taking up cancer cells. This process facilitated CTL activation via antigen presentation [4]. Moreover, Zhang et al. and Xiong et al. discovered that CD169 is a vital co-stimulatory molecule for activating CTLs in vitro [5, 6]. Based on these findings, several clinical studies have validated the role of CD169⁺ macrophages in cancer patients. Ohnishi et al., for example, identified a strong correlation between the number of CD169⁺ macrophages in lymph nodes (LNs) of colorectal cancer patients, the number of CTLs in tumor tissues, and the patients’ prognoses [7]. Comparable results have emerged from research on other types of cancer [8–13], underscoring the potential of effectively inducing CD169⁺ macrophages in vivo as a promising strategy for stimulating CTLs.

Several biomolecules capable of inducing CD169 expression have been identified, with interferon-alpha (IFNα) standing out due to its efficacy, mediated by the activation of signal transducer and activator of transcription (STAT) 1 and 2 [7, 13, 14]. Recent cancer immunotherapy targeting LNs has been focused on delivering stimulator of interferon genes (STING) agonist or Toll-like receptor 9 (TLR9) agonist to antigen-presenting cells for production of IFNα [15, 16]. However, the strategy to deliver IFNα directly to LN macrophages to induce CD169 more efficiently has not yet been realized. There are two pharmacokinetic challenges based on the molecular characteristics of IFNα that must be overcome to establish an effective IFNα delivery system to LN macrophages. The first challenge is the low LN drainage of IFNα, attributed to its molecular size of approximately 4.4 nm [17]. Generally, molecules sized between 5 nm and 100 nm can drain into lymphatic capillaries after subcutaneous injection [18], but the smaller size of IFNα may hinder its ability to enter these capillaries effectively. The second issue pertains to the targeting efficiency of IFNα to macrophages. The widespread expression of type I IFN receptors on various cells necessitates an elevated dose of IFNα to achieve the desired therapeutic effect on LN macrophages. However, increasing the dosage also elevates the risk of side effects, adversely affecting the patient’s quality of life and escalating medical costs. Therefore, drug delivery nano-technology is essential for harnessing IFNα’s potential in cancer immunotherapy, especially in inducing the CD169⁺ phenotype in LN macrophages.

Human serum albumin (HSA), the most plentiful protein in human blood, is distributed within intravascular and extravascular spaces. Approximately 40% of total HSA is found intravascularly while the remaining 60% is located in the extravascular space, which includes the interstitial and lymphatic fluids. Owing to its molecular size of approximately 10 nm, HSA efficiently drains into LNs after subcutaneous administration. This property has led researchers to explore the potential use of HSA as a diagnostic tool for detecting LN metastasis [19]. These insights have led us to propose a novel approach to overcoming the initial pharmacokinetic challenge associated with low LN drainage of IFNα by its conjugation with HSA. This hybrid would enhance the lymphatic delivery of IFNα due to the increased molecular size, overcoming a key barrier to utilizing IFNα effectively in cancer immunotherapy.

Wild-type HSA is a simple protein without glycan chains. To deliver drugs to macrophages, we had previously developed a mannosylated HSA (Man-HSA) attached with three mannosyl chains using the Pichia pastoris expression system [20]. We also established that Man-HSA and its polyethylene glycol derivative were preferentially distributed to Kupffer cells or tumor-associated macrophages, respectively, via the mannose receptors [21, 22]. These findings clarify that Man-HSA is a suitable carrier for targeting LN macrophages.

In the present study, we proposed a novel strategy of cancer immunotherapy, that is, “induction of the CD169⁺ phenotype in LN macrophages” based on the active targeting of IFNα to LN macrophages. For this purpose, we created the hybrid protein of mouse serum albumin with a highly mannosylated sugar chain (Man-MSA) and mouse IFNα (mIFNα) and subsequently clarified its potential as an “immune booster.” First, to confirm whether the CD169⁺ macrophage was a suitable target for cancer immunotherapy, whole-tissue imaging was conducted at single-cell resolution using LN samples from colorectal carcinoma patients. Then, Man-MSA-mIFNα was developed by albumin fusion technology. We thoroughly investigated its physicochemical and pharmacokinetic characteristics and assessed its capacity to induce CD169 expression both in vitro and in vivo. Moreover, the effects of Man-MSA-mIFNα on tumor growth and antitumor immunity were evaluated using several types of tumor-bearing mice. Finally, we also examined the combinational effect with an immune checkpoint inhibitor on tumor development. This study has the potential to provide the personalized medicine that tailors cancer immunotherapy based on the CD169⁺ macrophage content in LNs.

Multiplex tissue imaging and analysis

The procedure is similar to a previously described method [23]. Briefly, a formalin-fixed, paraffin-embedded block was sliced into 3 µm-thick sections, and a tissue section was mounted on a glass coverslip, deparaffinized by heating at 60°C for 20 min, and then submerging in xylene. The tissue was then rehydrated in a graded ethanol series. The sections were immersed in citrate buffer solution (10 mm, pH 6.0) and heated in a pressure cooker for antigen retrieval. After cooling to room temperature, the tissue was stained with antibodies for 3 hours at room temperature in a humidity chamber. After fixation with a post-staining fixing solution for 10 min, 100% methanol for 5 min, and final fixative solution for 20 min, the tissue was stored in PBS at 4 ℃ until image acquisition. Multiplex imaging was executed using a microfluidics instrument (Akoya Biosciences, Menlo Park, CA), a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan), and CODEX Instrument Manager software (Akoya Biosciences). An automated microfluidics system performed iterative annealing and stripping of fluorophore-labeled oligonucleotide barcodes complementary to the barcodes attached to antibodies. The raw image files were processed using CODEX processor version 1.8 (Akoya Biosciences). The antibodies and fluorophore-labeled oligonucleotide barcodes are described in Table S1.

QPTIFF files were imported to QuPath version 0.3.2. We extracted six 500 µm × 500 µm square regions in the image and performed cell segmentation on the basis of the nuclear stain using the cell detection algorithm in the software. To classify cells as being positive or negative for each marker, we generated training data for cell classification by deciding the positive and negative categories for every 10–30 cells in the regions based on fluorescence intensity. The results comprising the position (X,Y) and fluorescence intensity of each cell were exported as a csv file.

The csv file was imported to CytoMAP version 1.4.21. A detailed description of the workflow and functions built into CytoMAP is available in the online user manual. The representative area was subdivided into four regions by a 50 µm-radius raster-scanned neighborhoods function, which uses the Davies Bouldin Score and self-organizing map clustering methods, based on each fluorescence intensity. The analyses for neighborhood composition and Pearson correlation coefficients were performed based on the fluorescence intensity using the “Region Heatmaps” and “Cell-Cell Correlation” functions.

Preparation of fusion proteins

The designed fusion proteins are composed of MSA or MSA (D494N) linked to mIFNα2 (N78Q) via a polypeptide linker (-(Gly-Gly-Gly-Gly-Ser)₂-). In this study, we replaced asparagine at position 78 of mIFNα2 with glutamine (N78Q) to remove a glycosylation sequence originally present in mIFNα2. The flow chart describing the creation of the MSA (D494N)-mIFNα2 (N78Q) gene using pPIC9 is shown in Fig. S1. PCR was performed using a PfuTurbo DNA polymerase. To isolate the DNA fragment of the base sequence coding for MSA, restriction enzyme Xho1 and Ava1 recognition regions were inserted into the 5′ and 3′ terminals, respectively. Similarly, restriction enzyme Ava1 and EcoR1 recognition regions were inserted into the 5′ and 3′ terminals of the DNA sequence encoding mIFNα2. Then, pPIC9 was digested with Xho1 and EcoR1, and the appropriate pPIC9 fragment was extracted by agarose gel electrophoresis. To obtain the cDNA construct coding for MSA-mIFNα2, DNA fragments (pPIC9, MSA and mIFNα2) were ligated overnight at 16°C using a DNA ligation kit (Takara bio, Shiga, Japan). The subsequent mutation of MSA (D494N) and mIFNα2 (N78Q) was performed using a Quick Change kit (Takara bio) with the mutagenic primers described in Table S2. Hexa-His-tag was inserted into the C-terminal of mIFNα2 (N78Q), and Pichia pastoris (SMD1168 strain) was transformed with pPIC9-MSA (D494N)-mIFNα2 (N78Q) by electroporation according to the manual.

Pharmacokinetic analysis of ¹²⁵I-labeled proteins

The proteins were radiolabeled with ¹²⁵I according to previously reported procedures [24]. ¹²⁵I-labeled Man-MSA-mIFNα and MSA-mIFNα (1 mg protein kg^− 1; approximately 7 × 10⁶ counts per minute [CPM] per mouse), MSA (1 mg protein kg^− 1; approximately 6 × 10⁶ CPM per mouse), and mIFNα (1 µg protein kg^− 1; approximately 8 × 10⁵ CPM per mouse) were subcutaneously administered into the inguinal region of ICR mice. Inguinal LNs were isolated at each time point after the administration of the ¹²⁵I-labeled proteins, and the radioactivity was measured using an autowell gamma counter (AccuFlex γ7001; Hitachi Aloka Medical, Tokyo, Japan) according to the manufacturer's instructions.

Preparation of Cy5-labeled proteins

The lyophilized proteins were dissolved in deionized water to 20 mg mL^− 1. Cy5 NHS ester (0.5 mg) was added to the protein solution (1 mL), and the solution was mixed by rotation for 1 hour at room temperature. The unreacted Cy5 NHS ester was removed by ultrafiltration using an Amicon Ultra Ultracel-PL 10 kDa membrane filter (Merck Millipore, Burlington, MA).

Pharmacokinetic analysis of Cy5-labeled proteins

To evaluate the LN macrophage-targeting ability of Man-MSA-mIFNα, Cy5-labeled fusion proteins (60 nmol kg^− 1) were subcutaneously administered into the inguinal region of ICR mice, and the inguinal LNs were isolated 1 hour after administration.

Flow cytometry: LNs or tumors were harvested, minced finely with scissors on ice, and then incubated with collagenase D (Sigma-Aldrich) or Dri tumor & tissue dissociation reagent (Becton Dickinson, NJ) at 37°C for 30 min. After passing the digested mixture through a 70 µm filter, the cells were incubated with ACK lysis buffer (NH₄Cl [0.15 m], KHCO₃ [10 mm], and EDTA [0.1 mm]) to remove red blood cells and suspended in FACS buffer (EDTA/PBS [2 mm] containing 0.5% FBS). The cells were incubated with FcR blocking reagent (1:50, Miltenyi Biotec, 130-092-575) and Fixable Viability Dye eFluor 780 (1:5,000, Invitrogen, 65-0865-14) at room temperature for 5 min and then, stained with antibodies for cell surface markers at 4°C for 15 min. After washing with FACS buffer, the cells were fixed, permeabilized, and incubated with antibodies for intracellular markers at 4°C for 30 min. The cells were analyzed on a FACSVerse (Becton Dickinson) flow cytometer with FACSuite (Becton Dickinson) software. The data were analyzed using FlowJo software (Becton Dickinson). The gating strategies are shown in Fig. S2 and Fig. S3, and the antibodies in Table S1.

Evaluation of CD169 expression in vitro

J774.1 cells were seeded at a density of 3.0 × 10⁵ cells per well in 12-well plates and incubated overnight at 37°C before the addition of mIFNα (1 IU mL^− 1), MSA, MSA-mIFNα, or Man-MSA-mIFNα (70 nm) to the cells. The cells were collected after incubation for 24 hours at 37°C, and CD169 expression was evaluated by flow cytometry.

Evaluation of CD169 expression in vivo

We first assessed the optimal dose of Man-MSA-mIFNα and the time course of CD169 expression. As shown in Fig. S4 and Fig. S5, we confirmed that, 72 hours after administration, Man-MSA-mIFNα (0.6 nmol kg^− 1) showed high CD169 expression on LN macrophages. Next, to compare the in vivo CD169 induction levels of mIFNα, MSA-mIFNα and Man-MSA-mIFNα, we evaluated the titer of mIFNα using J774.1 cells and found the induction level of CD169 after treatment with mIFNα (1 IU mL^− 1) to be equivalent to that after Man-MSA-mIFNα (6 µg mL^− 1, 0.07 nmol mL^− 1) (Fig. S6). Thus, mIFNα (8 IU kg^− 1), MSA-mIFNα (0.6 nmol kg^− 1), or Man-MSA-mIFNα (0.6 nmol kg^− 1) was subcutaneously administered into the inguinal region of C57BL/6N mice. Seventy-two hours after administration, inguinal LNs were harvested, and the CD169 expression on macrophages evaluated by flow cytometry.

Antitumor effects of Man-MSA-mIFNα

We first evaluated the optimal dosing interval using MB49-bearing mice and found that the administration of Man-MSA-mIFNα once every 4 days, but not once every 7 days, exerted significant antitumor effects (Fig. S7). Therefore, mIFNα (8 IU kg^− 1), MSA-mIFNα, and Man-MSA-mIFNα (0.6 nmol kg^− 1) were injected subcutaneously near the tumor once every 4 days. For combination therapy, the mice were treated intraperitoneally with isotype control antibodies (200 µg; BioLegend, 400667) and anti-PD-L1 antibodies (200 µg; BioLegend, 124329) on the 10th and 13th days after tumor inoculation and then, subcutaneously with Man-MSA-mIFNα (0.3 nmol kg^− 1) once every 4 days. The tumor volume was calculated according to the formula V = 0.4 × a × b², where “a” is the major axis, and “b” is the minor axis of the tumor. Tumor-bearing mice were sacrificed on the 18th day after tumor inoculation.

Statistical analysis

The experimental data were analyzed using GraphPad Prism 9. The means for two-group data were compared by using an unpaired t-test, and the means for more than two groups were compared by one-way ANOVA followed by Tukey’s multiple comparison or Bonferroni's multiple comparison. The probability value of p < 0.05 was considered to be a statistically significant difference.

Spatial identification of LN cells in cancer patients

To confirm whether CD169⁺ macrophage-mediated T cell priming in mouse LNs also occurs in LNs of cancer patients [4], we first predicted cell-cell interactions with critical physiological roles at both cellular and tissue levels by obtaining positional information about individual cells (macrophages, CD8⁺ and CD4⁺ T cells, B cells, and vascular endothelial cells). This information was obtained by applying a single-cell and spatial multi-omics system, the Akoya PhenoCycler system [23], to regional LN biopsies of colorectal carcinoma patients. As shown in Fig. 1A, we immunostained a regional LN of a colorectal carcinoma patient with anti-CD68 (macrophage), anti-CD8 (CD8⁺ T cell), anti-CD4 (CD4⁺ T cell), anti-CD20 (B cell), and anti-CD34 (vascular endothelial cell) antibodies and visualized each cell in the LN sinuses, which contain an abundance of CD169⁺ macrophages (Fig. 1B) [7, 25]. We next imported the fluorescence intensity and positional information (X, Y) of individual cells to CytoMAP, a spatial analytics platform that incorporates diverse statistical and visualization modules [26]. The tissue images (500 × 500 µm) were subdivided into 50 µm-radius neighborhoods, which were mapped based on the cellular composition. Four regions with different cellular compositions were defined using the “raster scanned neighborhoods” function (Fig. 1C). We further visualized the mean channel intensities per neighborhood with heatmaps and found that each region has the following cellular characteristics: Region 1, mixed cellular composition (macrophages, CD8⁺ and CD4⁺ T cells, B cells, vascular endothelial cells) with low cell density; Region 2, B cell-rich composition; Region 3, macrophage- and CD8⁺ and CD4⁺ T cell-rich composition; Region 4, mixed cellular composition (macrophages, CD8⁺ and CD4⁺ T cells, B cells, vascular endothelial cells) with high cell density (Fig. 1D and Fig. S8A). In addition, Pearson correlation coefficients of the number of cells per neighborhood showed that the number of macrophages positively correlated with the number of CD8⁺ (r = 0.57) and CD4⁺ T cells (r = 0.76) in the neighborhoods (Fig. 1E and Fig. S8B). These results indicate that there were regions where CD169⁺ macrophages may prime T cells within the regional LNs of cancer patients.

Lymphatic drainage of cancer antigens in tumor-bearing mice

The effective activation of CTLs via antigen presentation is dependent not only on the co-stimulatory signals presented by CD169 but also on the engulfment of antigens by CD169⁺ macrophages. To examine whether cancer antigens from tumor tissues drain into LNs and get engulfed by CD169⁺ macrophages, we used anti-tyrosinase related protein-2 (TRP-2, a melanoma-associated self-antigen) and anti-CD169 antibodies to immunostain the inguinal LNs of tumor-bearing mice that had been subcutaneously injected with B16F10 melanoma cells in the inguinal area [27]. The fluorescence derived from anti-TRP-2 antibodies was observed in the tumor-draining LNs (tdLNs) but barely detectable in the non-tumor-draining LNs (ndLNs) and spleens of the tumor-bearing mice (Fig. 1F and Fig. S9). Enlarged images of the tdLN revealed that this fluorescence co-localized with the fluorescence derived from anti-CD169 antibodies in the sinus areas rich in CD169⁺ macrophages (Fig. 1G).

Expression of mannose receptors and type Ⅰ IFN receptors in LN macrophages

To verify whether subcutaneously administered Man-HSA is distributed to macrophages in the LN sinuses, we first examined CD206 (mannose receptor) expression in the regional LNs of a colorectal carcinoma patient. Combined staining with hematoxylin-eosin (H.E.) and immunohistochemistry with antibodies against CD163 (a marker for LN sinus macrophages) and CD206 identified the sinus [25], which is marked with an asterisk, and found high expression of CD206 and CD163 in subcapsular and medullary LN sinuses (Fig. S10A). Furthermore, triple immunostaining with antibodies for CD68, CD206, and interferon alpha/beta receptor 1 (IFNAR1) showed that macrophages in LN sinuses expressed both CD206 and IFNAR1 (Fig. S10B).

Lymphatic drainage of albumin in mice

To investigate whether albumin possesses characteristics for lymphatic drainage, we subcutaneously administered radioiodine (¹²⁵I)-labeled MSA or mIFNα to the inguinal region of mice. More than 20% of the initial dose (ID) per tissue (g) of MSA was distributed in inguinal LN an hour after the administration, whereas the distribution was lower in the case of mIFNα (Fig. S11). We also observed a marked reduction in radioactivity in the mIFNα-administration group at 6 hours after the administration. Once the ¹²⁵I-labeled proteins are taken up into cells through receptor-mediated endocytosis and degraded in lysosomes, the radio-catabolites are rapidly excreted from the cells [28]. Therefore, mIFNα was supposed to be taken up by the type I IFN receptor-expressing cells and degraded within 6 hours after the administration [29]. These results suggest that HSA may improve lymphatic drainage of IFNα. Therefore, we attempted to develop a genetically fused chimeric protein of mIFNα and Man-MSA because the IFN activity, albumin kinetics, and immunogenic response are largely dependent on animal species [30–32].

Design and physicochemical properties of Man-MSA-mIFNα

We had previously developed Man-HSA by inserting three N-glycation sequences (D63N, A320T, and D494N) into HSA [20]. Analysis using matrix-assisted laser desorption ionization-time of flight-mass spectrometry revealed that the D63N variant had 0–1 mannose residues, A320T had 1–2, and D494N had 11 mannose residues, indicating that D494N was largely responsible for the mannosylation [20]. The clustering effect of highly mannosylated structures plays a crucial role in binding to the mannose receptor [33]. To obtain highly mannosylated forms of the protein while minimizing the mutations, the present study fused His-tag-conjugated mIFNα with mutated MSA (Man-MSA) whose aspartic acid (D) at position 494 was replaced with asparagine (D494N) (Fig. 2A). We first performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified fusion proteins to confirm the fusion of mIFNα and mutated MSA. Coomassie Brilliant Blue (CBB) staining showed that the position of the Man-MSA-mIFNα band was slightly higher than that of MSA-mIFNα, but both bands corresponded to the theoretical molecular weight (Fig. 2B). The presence of mannose chains in Man-MSA-mIFNα was further corroborated through Periodic acid-Schiff (PAS) staining. PAS stain reacted positively with Man-MSA-mIFNα and α1-acid glycoprotein (AGP), while such a positive reaction was not observed in the case of MSA-mIFNα (Fig. 2C). Western blotting analyses were carried out using anti-MSA and anti-His-tag antibodies to confirm the presence of MSA and His-tag-conjugated mIFNα in the fusion protein. Man-MSA-mIFNα and MSA-mIFNα reacted positively with both antibodies, but MSA positively reacted with only anti-MSA antibodies (Fig. 2D, E). These data indicate that Man-MSA-mIFNα was formed from the fusion of the intact Man-MSA and mIFNα. We further analyzed the secondary and tertiary structures of Man-MSA-mIFNα by circular dichroism (CD) spectroscopy. As shown in Fig. 2F, G, the CD spectrum of Man-MSA-mIFNα was similar to that of MSA, indicating that Man-MSA largely retained the characteristic structure of MSA despite fusion with mIFNα.

Intralymph node distribution of Man-MSA-mIFNα and its capacity to induce the CD169⁺ phenotype of macrophages

To evaluate the lymphatic drainage of Man-MSA-mIFNα, we performed a similar experiment as in Fig. S11. Similar to MSA alone, over 20% of the ID per tissue (g) of both Man-MSA-mIFNα and MSA-mIFNα were distributed in the LN (Fig. 3A). Interestingly, a notable decline in radioactivity in both the MSA-mIFNα- and Man-MSA-mIFNα-administration groups was observed 6 hours after the administration, indicating that they were taken up by the type I IFN receptor- or mannose receptor-expressing cells in the LNs [29, 34]. We additionally measured the distribution of Man-MSA-mIFNα in the main organs (heart, liver, kidney, lung, and spleen) and found that Man-MSA-mIFNα showed remarkably low percentages of the ID per tissue (g) compared to that observed in the inguinal LN, indicating poor distribution in the main organs (Fig. S12). To further investigate whether Man-MSA-mIFNα has the capability to target macrophages, we subcutaneously administered Cy5-labeled Man-MSA-mIFNα to mice and immunostained the LNs with anti-CD169 and anti-CD11b antibodies. An hour after the administration, fluorescence associated with Cy5-labeled Man-MSA-mIFNα and MSA-mIFNα was detected in the LN sinuses (Fig. 3B). A more careful observation found that the co-localization of Cy5-labeled Man-MSA-mIFNα with CD169⁺ and CD11b⁺ macrophages was markedly higher than that of MSA-mIFNα (Fig. 3C, D). In contrast, the co-localization of Man-MSA-mIFNα with CD31⁺ lymphatic endothelial cells, which are components of the sinus, was significantly lower than that of MSA-mIFNα (Fig. S13).

To evaluate the ability of Man-MSA-mIFNα to induce the CD169⁺ phenotype, the expression of CD169 was analyzed by flow cytometry 24 hours after the treatment of a mouse macrophage cell line, i.e., J774.1, with Man-MSA-mIFNα, MSA-mIFNα, or mIFNα. As we expected, Man-MSA-mIFNα, MSA-mIFNα, and mIFNα significantly induced CD169 expression in the cells (Fig. 3E). We then administered Man-MSA-mIFNα subcutaneously to mice and evaluated CD169 expression in LN macrophages. Even though the titers of mIFNα, MSA-mIFNα, and Man-MSA-mIFNα were set to be equal (Fig. S6), Man-MSA-mIFNα resulted in significantly higher CD169 expression than both mIFNα and MSA-mIFNα after 72 hours post administration (Fig. 3F). These data demonstrate that Man-MSA-mIFNα efficiently induced the CD169⁺ phenotype in LN macrophages due to its high lymphatic drainage and macrophage-targeting ability.

Antitumor effect of Man-MSA-mIFNα in tumor-bearing mice

It is well known that immunogenic tumors are characterized by high T cell infiltration. In contrast, such infiltration is not observed in the case of non-immunogenic tumors [35]. To identify the types of cancers that Man-MSA-mIFNα could potentially be useful against, we examined the antitumor effects of Man-MSA-mIFNα on mice inoculated with a mouse bladder carcinoma cell line (MB49; a non-immunogenic tumor) or a mouse colon adenocarcinoma cell line (MC38; an immunogenic tumor) (Fig. 4A) [36, 37]. Man-MSA-mIFNα exerted significant antitumor effects against MB49-bearing mice, accompanied by an induction of CD169 expression in the LNs (Fig. 4B); however, no such effect was observed in MC38-bearing mice (Fig. 4C). Similar antitumor impacts of Man-MSA-mIFNα were observed in mice bearing the murine Lewis lung carcinoma cell line (LLC; a non-immunogenic tumor) (Fig. 4D) [38]. Next, we compared the antitumor effects of Man-MSA-mIFNα with other mIFNα formulations using MB49-bearing mice. Consistent with the CD169 expression level shown in Fig. 3F, Man-MSA-mIFNα significantly inhibited tumor growth compared to mIFNα and MSA-mIFNα on the 18th day after tumor inoculation (Fig. 4E, F). Since the expression levels of CD169 contribute to the activation of CTLs, we further examined CTL activation in LNs and the number of CTLs in tumor tissue. As expected, Man-MSA-mIFNα significantly increased the number of CD8⁺ T cells and boosted the expression levels of Ki67 and the production of IFNγ and CD69, both markers indicative of CTL activation, in LNs (Fig. 4G). Consistent with the CTL activation in LNs, Man-MSA-mIFNα substantially enhanced the infiltration of CD8⁺ T cells into the tumor (Fig. 4H).

Antitumor effect of Man-MSA-mIFNα in CD169⁺ macrophage-depleted mice

To demonstrate whether the antitumor effect of Man-MSA-mIFNα is dependent on LN CD169⁺ macrophages, we used CD169-DTR mice in which LN CD169⁺ macrophages can be depleted by the subcutaneous injection of diphtheria toxin (DT) (Fig. 5A) [4]. Under the conditions where the number of CD169⁺ macrophages in the LNs was significantly lowered by DT injection (Fig. 5B), the administration of Man-MSA-mIFNα did not decrease the tumor volume and weight of MB49-bearing CD169-DTR mice compared to those of the saline-treated group (Fig. 5C, D). In addition, Man-MSA-mIFNα treatment also did not affect CTL activation and infiltration in tumor tissues in CD169-DTR mice (Fig. 5E, F). These findings show that the antitumor effects of Man-MSA-mIFNα were mediated by its activation of CTLs via the induction of the CD169⁺ phenotype of LN macrophages.

Antitumor effect of a combination therapy of Man-MSA-mIFNα and PD-L1 blockade

Since the remarkable antitumor effects of immune checkpoint inhibitors are attributed to the suppression of CTL exhaustion signaling, these treatments may not be as beneficial for cancer patients exhibiting minimal CTL infiltration into tumors [39]. However, Man-MSA-mIFNα has demonstrated an ability to boost antitumor immunity and increase CTL tumor infiltration substantially. Consequently, we speculated that the combination of Man-MSA-mIFNα and immune checkpoint inhibitors could ameliorate treatment resistance in patients who are unresponsive to immune checkpoint inhibitors. To confirm this hypothesis, we evaluated the antitumor effects of a combination therapy using MB49-bearing mice, which are known to exhibit resistance to PD-L1 blockade monotherapy (Fig. 6A) [40]. As expected, no significant treatment effect was observed with the PD-L1 blockade alone (Fig. 6B, C). In contrast, the combination therapy showed a significant antitumor effect compared to the PD-L1 blockade or Man-MSA-mIFNα alone. Consistent with these results, we observed a decrease in Ki67⁺ cells, a marker for tumor growth, and an increase in apoptotic cells in the tumor tissues after the combination therapy (Fig. 6D). Furthermore, the number of activated CTLs in the tumor tissue showed a significant increase following combination therapy (Fig. 6E). Taken together, these results suggest that Man-MSA-mIFNα functioned as an “immune booster” that attenuated therapeutic resistance to immune checkpoint inhibitors by increasing CTL tumor infiltration.

Safety evaluation

Given that the administration of IFNα induces a variety of side effects such as gastrointestinal symptoms, leukopenia, thrombocytopenia, anemia, liver damage, and renal damage [41], there are concerns that Man-MSA-mIFNα administration might induce similar adverse reactions. To investigate this possibility, we assessed several IFNα-associated side effects in tumor-bearing mice that had been treated with Man-MSA-mIFNα. As shown in Fig. S14A, no body weight loss or reduction in white blood cells (WBCs) and platelets (PLTs) was observed in any treatment group. In addition, similar results were obtained for red blood cells (RBCs), hemoglobin (HGB), and the hematocrit value (HCT). The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) showed no abnormal values in any group. H.E. staining also revealed no tissue damage (Fig. S14B). These findings suggest that, under our experimental conditions where significant antitumor effects were observed in the Man-MSA-mIFNα-treated group (7-week-old male C57BL/6N mice), Man-MSA-mIFNα is unlikely to elicit side effects related to IFNα, such as weight loss, bone marrow suppression, or organ damage.

Lymphatic drainage of subcutaneously administered substances is regulated by molecular size (5–100 nm), molecular weight (> 20 kDa), and zeta potential (negatively charged surfaces) [42]. Interestingly, albumin, which is widely used as a drug delivery system (DDS) carrier for prolonging the circulation time of drugs and proteins, is also qualified as a LN-targeted DDS carrier due to its fascinating molecular properties—10 nm (diameter), 66.5 kDa (molecular mass), and negatively charged surface. Our pharmacokinetic experiments using radioactive iodine demonstrated that albumin-fused mIFNα gained ~ 1.6 times greater access to a LN than mIFNα alone (Fig. S11 and Fig. 3A). Interstitial oncotic pressure also contributes to lymphatic drainage. Trubetskoy et al. found that massaging the injection site of liposomes enhanced the lymphatic absorption of liposomes [43]. Albumin also increases the interstitial pressure at the injection site due to its osmoregulatory function. In fact, subcutaneously administered HSA mixed with IFNα improves the lymphatic absorption of the IFNα [44]. Based on these unique properties, it is reasonable to infer that albumin is a suitable DDS carrier to target LNs.

In the present study, we successfully applied Man-MSA as a DDS to target LN macrophages and induced the CD169⁺ phenotype of macrophages by delivering IFNα to the cells (Fig. 3). Resident conventional dendritic cells (cDCs) in LNs also contribute to CTL activation via antigen presentation [45]. The IFNα response further enhances their functions through the increased expression of CD80/86 (co-stimulatory molecules) and major histocompatibility complex class I. Given that DCs, like macrophages, express mannose and type I IFN receptors, it is possible that the antitumor effect of Man-MSA-mIFNα resulted from an off-target effect on cDCs. However, it is unlikely that Man-MSA-mIFNα would distribute to cDCs because of the structural features of LNs. LNs are structurally classified into the cortex, paracortex, and medulla. CD169⁺ macrophages are located in the subcapsular (cortex) and medullary (medulla) sinuses adjacent to afferent and efferent lymphatics [46]. In contrast, cDCs are located in the paracortex, deep in the LNs [47]. To gain access deep in the LN, lymph-borne molecules must flow through the conduit, branching out from the lymphatic sinuses. Since the conduit is very narrow (3–5 nm), only molecules smaller than 70 kDa can reach deep inside the LN [48]. Our results showed that Cy5-labeled Man-MSA-mIFNα (88.5 kDa) localized in the subcapsular and medullary sinuses of a LN with few cDCs and abundant CD169⁺ macrophages (Fig. 3B). These data corroborate the fact that not only is Man-MSA highly mannosylated but it also has the desired molecular size to target LN macrophages.

Asano et al. subcutaneously administered dead cancer cells to mice and found that they were taken up by CD169⁺ macrophages in the LNs [4]. In the present study, we evaluated the distribution of cancer antigens in the LNs of B16F10 tumor-bearing model mice to monitor the movement of cancer antigens under conditions mimicking the biological environment. Cancer antigens were not observed in the spleen, but in the regional LN, they were colocalized with CD169⁺ macrophages (Fig. 1F, G). Furthermore, as mentioned above, the fusion of Man-MSA and mIFNα improved the distribution of mIFNα to LNs and CD169⁺ macrophages and efficiently induced CD169 expression (Fig. 3). These results show that LN CD169⁺ macrophages internalized cancer antigens that had been distributed to the LNs, and the subsequent process of antigen presentation to T cells was likely boosted by Man-MSA-mIFNα. Here, to achieve the antitumor effect of Man-MSA-mIFNα, it must be injected subcutaneously near the tumor and delivered to LN macrophages exposed to the cancer antigens. However, given the wide variety of carcinomas, it is challenging to administer drugs subcutaneously in the vicinity of the tumor in an actual clinical setting. Therefore, in the future, it may be necessary to broaden the subcutaneous administration sites from 'near the tumor' to 'throughout the body' by incorporating not only mIFNα but also cancer antigens into Man-MSA.

In this study, we prepared tumor-bearing mice with immunogenic tumors (MC38) or non-immunogenic tumors (MB49 and LLC), each exhibiting different levels of tumor immunogenicity. Interestingly, Man-MSA-mIFNα treatment exerted a significant antitumor effect and remarkably enhanced the CD169⁺ phenotype in LN macrophages only in non-immunogenic tumor-bearing mice (Fig. 4B-D). CD169 expression has been used as a surrogate biomarker for type I interferon activity in many diseases [49]. Therefore, we speculate that the difference in the antitumor effect of Man-MSA-mIFNα between immunogenic and non-immunogenic tumors was due to the varying endogenous IFNα expression levels in these two types of cancer. In other words, the CD169 expression levels of LN macrophages would be highly regulated in immunogenic tumors with high endogenous IFNα levels. As a result, Man-MSA-mIFNα failed to induce the CD169⁺ phenotype of macrophages in LNs, not yielding any antitumor effect. In the clinical setting, the numbers of CD169⁺ macrophages in LNs are known to be different in patients even with the same cancer type [25]. Therefore, our IFNα formulation could potentially be beneficial for cancer patients pathologically diagnosed with low CD169 expression levels via LN biopsy. In the future, it will be necessary to develop non-invasive methods for monitoring LN CD169, such as liquid biopsy for IFNα, to tailor individual cancer immunotherapy based on the CD169⁺ macrophage content.

The present study successfully achieved our therapeutic strategy of "the induction of the CD169⁺ phenotype in LN macrophages" by active targeting of IFNα to LN macrophages. Therefore, it can be expected that this cancer immunotherapy allows for the combination of Man-MSA-mIFNα with not only immune checkpoint inhibitors but also other immune-enhancing drugs, such as CAR-T therapy, radiotherapy, and anticancer drugs, for the treatment of intractable cancers that are challenging to treat with single agents or existing combination therapies. Further, we previously reported the development of a genetic fusion of human IFNα and mannosylated human serum albumin (Man-HSA-hIFNα) [50]. The present study demonstrated that Man-HSA-hIFNα induces the CD169⁺ phenotype in human monocyte-derived macrophages (Fig. S15). Therefore, Man-HSA-hIFNα, optimized through our drug discovery technologies, is expected to exert a significant antitumor effect in humans.

The present study successfully designed and developed Man-MSA-mIFNα, which efficiently induces the CD169⁺ phenotype in LN macrophages, contributing to the antitumor immune response. The findings of this study not only reveal the usefulness of a therapeutic strategy targeting CD169⁺ macrophages in the LNs but also suggest that Man-HSA-hIFNα could be a promising immune booster for cancer patients who are unresponsive to immune checkpoint inhibitors.

AGP: α1-acid glycoprotein

ALT: alanine aminotransferase

AST: aspartate aminotransferase

BUN: blood urea nitrogen

CBB: Coomassie Brilliant Blue

CD: circular dichroism

cDC: conventional dendritic cell

CTL: cytotoxic T lymphocyte

DDS: drug delivery system

DT: diphtheria toxin

GMFI: geometric mean fluorescence intensity

HCT: hematocrit

H.E.: hematoxylin-eosin

HGB: hemoglobin

ID: initial dose

IFN: interferon

IFNAR1: interferon alpha/beta receptor 1

LLC: Lewis lung carcinoma

LN: lymph node

Man-HSA: mannosylated human serum albumin

Man-MSA: mannosylated mouse serum albumin

mIFNα: mouse interferon alpha

ndLN: non-tumor-draining LN

PAS: Periodic acid-Schiff

PD-1: programmed death-1

PD-L1: programmed death-ligand 1

PLT: platelet

RBC: red blood cell

s.c.: subcutaneous

SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis

tdLN: tumor-draining LN

TRP-2: tyrosinase related protein-2

TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling

WBC: white blood cell

¹²⁵I: radioiodine

Ethics approval and consent to participate

The study design was approved by the Institutional Review Board of Kumamoto University (#1169) in accordance with the guidelines for Good Clinical Practice and the Declaration of Helsinki. Paraffin-embedded LN samples were prepared from specimens obtained from patients diagnosed with colorectal cancer that were surgically resected at Izumi General Hospital (Izumi, Kagoshima, Japan). Written informed consent was obtained from all patients, and the study design was approved by the review board (#57). The need for individual patient consent for inclusion in the study was waived by the Institutional Review Board of Kumamoto University (#1169). All animal experiments were conducted using procedures approved by the experimental animal ethics committee at Kumamoto University (A2021-021).

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was partially supported by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Research Fellow (JP23KJ1774), a Grant-in-Aid for Scientific Research (B) (JP23K06362), a Grant-in-Aid for Young Scientists (B) (JP21K15315), a Grant-in-Aid for Scientific Research (B) (JP20H03406), a Grant-in-Aid for Scientific Research (B) (JP20H03459), and a Grant-in-Aid for Scientific Research (B) (JP23H03742) from JSPS.

Author contributions

R.F., Y.F., H.M., and T.M. conceived the work. R.F., Y.F., H.M., H.W., Y.K., and T.M. designed the research studies. R.F., H.M., C.P., Y.M. (Yuki Minayoshi), H.Y., Y.M. (Yuki Mizuta), M.T., R.Y., and Y.S. conducted experiments and acquired data. K.H., S.I., K.Y., K.O., and J.S. contributed new reagents or analytic tools. R.F., Y.F., H.M., Y.K., and T.M. wrote the paper. K.O., J.S., M.O., and H.W. critically reviewed the data and manuscript. Y.F., H.M., H.W., Y.K., and T.M. supervised the project. All authors read and approved the final manuscript.

Acknowledgements

The authors wish to thank Megan Pavlak for assisting with the modification of the manuscript.

Yang Y, Attwood K, Bshara W, Mohler JL, Guru K, Xu B, Kalinski P, Chatta G. High intratumoral CD8(+) T-cell infiltration is associated with improved survival in prostate cancer patients undergoing radical prostatectomy. Prostate. 2021;81:20-28.
Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science. 2018;359:1350-1355.
Mellman I, Chen DS, Powles T, Turley SJ. The cancer-immunity cycle: Indication, genotype, and immunotype. Immunity. 2023;56:2188-2205.
Asano K, Nabeyama A, Miyake Y, Qiu CH, Kurita A, Tomura M, Kanagawa O, Fujii S, Tanaka M. CD169-positive macrophages dominate antitumor immunity by crosspresenting dead cell-associated antigens. Immunity. 2011;34:85-95.
Zhang Y, Li JQ, Jiang ZZ, Li L, Wu Y, Zheng L. CD169 identifies an anti-tumour macrophage subpopulation in human hepatocellular carcinoma. J Pathol. 2016; 239:231-241.
Xiong YS, Wu AL, Lin QS, Yu J, Li C, Zhu L, Zhong RQ: Contribution of monocytes Siglec-1 in stimulating T cells proliferation and activation in atherosclerosis. Atherosclerosis. 2012;224:58-65.
Ohnishi K, Komohara Y, Saito Y, Miyamoto Y, Watanabe M, Baba H, Takeya M. CD169-positive macrophages in regional lymph nodes are associated with a favorable prognosis in patients with colorectal carcinoma. Cancer Sci. 2013;104:1237-1244.
Asano T, Ohnishi K, Shiota T, Motoshima T, Sugiyama Y, Yatsuda J, Kamba T, Ishizaka K, Komohara Y. CD169-positive sinus macrophages in the lymph nodes determine bladder cancer prognosis. Cancer Sci. 2018;109:1723-1730.
Björk Gunnarsdottir F, Auoja N, Bendahl PO, Rydén L, Fernö M, Leandersson K. Co-localization of CD169(+) macrophages and cancer cells in lymph node metastases of breast cancer patients is linked to improved prognosis and PDL1 expression. Oncoimmunology. 2020;9:1848067.
Kumamoto K, Tasaki T, Ohnishi K, Shibata M, Shimajiri S, Harada M, Komohara Y, Nakayama T. CD169 Expression on Lymph Node Macrophages Predicts in Patients With Gastric Cancer. Front Oncol. 2021;11:636751.
Ohnishi K, Yamaguchi M, Erdenebaatar C, Saito F, Tashiro H, Katabuchi H, Takeya M, Komohara Y. Prognostic significance of CD169-positive lymph node sinus macrophages in patients with endometrial carcinoma. Cancer Sci. 2016;107:846-852.
Takeya H, Shiota T, Yagi T, Ohnishi K, Baba Y, Miyasato Y, Kiyozumi Y, Yoshida N, Takeya M, Baba H, Komohara Y. High CD169 expression in lymph node macrophages predicts a favorable clinical course in patients with esophageal cancer. Pathol Int. 2018; 68:685-693.
Saito Y, Ohnishi K, Miyashita A, Nakahara S, Fujiwara Y, Horlad H, Motoshima T, Fukushima S, Jinnin M, Ihn H, et al. Prognostic Significance of CD169+ Lymph Node Sinus Macrophages in Patients with Malignant Melanoma. Cancer Immunol Res. 2015; 3:1356-1363.
Rincon-Arevalo H, Aue A, Ritter J, Szelinski F, Khadzhynov D, Zickler D, Stefanski L, Lino AC, Körper S, Eckardt KU, et al. Altered increase in STAT1 expression and phosphorylation in severe COVID-19. Eur J Immunol. 2022;52:138-148.
Chen F, Li T, Zhang H, Saeed M, Liu X, Huang L, Wang X, Gao J, Hou B, Lai Y, et al. Acid-Ionizable Iron Nanoadjuvant Augments STING Activation for Personalized Vaccination Immunotherapy of Cancer. Adv Mater. 2023;35:e2209910.
Hu J, Xu J, Li M, Zhang Y, Yi H, Chen J, Dong L, Zhang J, Huang Z. Targeting Lymph Node Sinus Macrophages to Inhibit Lymph Node Metastasis. Mol Ther Nucleic Acids. 2019;16:650-662.
Wang G, Hu J, Gao W. Tuning the molecular size of site-specific interferon-polymer conjugate for optimized antitumor efficacy. Science China Materials. 2017;60:563-570.
Moyer TJ, Zmolek AC, Irvine DJ. Beyond antigens and adjuvants: formulating future vaccines. J Clin Invest. 2016;126:799-808.
Valhondo-Rama R, Sánchez Izquierdo N, Tapias Mesa A, Perissinotti A, Vidal-Sicart S. Comparison of 99mTc-Tilmanocept and Hybrid Indocyanine Green-99mTc-Albumin Nanocolloid Drainage in a Patient With Melanoma in the Scalp. Clin Nucl Med. 2020;45:977-979.
Hirata K, Maruyama T, Watanabe H, Maeda H, Nakajou K, Iwao Y, Ishima Y, Katsumi H, Hashida M, Otagiri M. Genetically engineered mannosylated-human serum albumin as a versatile carrier for liver-selective therapeutics. J Control Release. 2010;145:9-16.
Maeda H, Hirata K, Watanabe H, Ishima Y, Chuang VT, Taguchi K, Inatsu A, Kinoshita M, Tanaka M, Sasaki Y, et al. Polythiol-containing, recombinant mannosylated-albumin is a superior CD68+/CD206+ Kupffer cell-targeted nanoantioxidant for treatment of two acute hepatitis models. J Pharmacol Exp Ther. 2015;352:244-257.
Mizuta Y, Maeda H, Ishima Y, Minayoshi Y, Ichimizu S, Kinoshita R, Fujita I, Kai T, Hirata K, Nakamura T, et al. A Mannosylated, PEGylated Albumin as a Drug Delivery System for the Treatment of Cancer Stroma Cells. Advanced Functional Materials. 2021;31:2104136.
Black S, Phillips D, Hickey JW, Kennedy-Darling J, Venkataraaman VG, Samusik N, Goltsev Y, Schürch CM, Nolan GP. CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nat Protoc. 2021;16:3802-3835.
Watanabe H, Yamasaki K, Kragh-Hansen U, Tanase S, Harada K, Suenaga A, Otagiri M. In vitro and in vivo properties of recombinant human serum albumin from Pichia pastoris purified by a method of short processing time. Pharm Res. 2001;18:1775-1781.
Komohara Y, Ohnishi K, Takeya M. Possible functions of CD169-positive sinus macrophages in lymph nodes in anti-tumor immune responses. Cancer Sci. 2017; 108:290-295.
Stoltzfus CR, Filipek J, Gern BH, Olin BE, Leal JM, Wu Y, Lyons-Cohen MR, Huang JY, Paz-Stoltzfus CL, Plumlee CR, et al. CytoMAP: A Spatial Analysis Toolbox Reveals Features of Myeloid Cell Organization in Lymphoid Tissues. Cell Rep. 2020; 31:107523.
Takeshima T, Chamoto K, Wakita D, Ohkuri T, Togashi Y, Shirato H, Kitamura H, Nishimura T. Local radiation therapy inhibits tumor growth through the generation of tumor-specific CTL: its potentiation by combination with Th1 cell therapy. Cancer Res. 2010;70:2697-2706.
Cilliers C, Liao J, Atangcho L, Thurber GM. Residualization Rates of Near-Infrared Dyes for the Rational Design of Molecular Imaging Agents. Mol Imaging Biol. 2015; 17:757-762.
Zanin N, Viaris de Lesegno C, Lamaze C, Blouin CM. Interferon Receptor Trafficking and Signaling: Journey to the Cross Roads. Front Immunol. 2020;11:615603.
Krieckaert CL, Bartelds GM, Wolbink GJ. Therapy: Immunogenicity of biologic therapies-we need tolerance. In Nat Rev Rheumatol. Volume 6. United States2010;558-559.
Nilsen J, Bern M, Sand KMK, Grevys A, Dalhus B, Sandlie I, Andersen JT. Human and mouse albumin bind their respective neonatal Fc receptors differently. Sci Rep. 2018;8:14648.
Jacobs S, Wavreil F, Schepens B, Gad HH, Hartmann R, Rocha-Pereira J, Neyts J, Saelens X, Michiels T. Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ. J Interferon Cytokine Res. 2018;38:469-479.
McGreal EP, Rosas M, Brown GD, Zamze S, Wong SY, Gordon S, Martinez-Pomares L, Taylor PR. The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose. Glycobiology. 2006;16:422-430.
van der Zande HJP, Nitsche D, Schlautmann L, Guigas B, Burgdorf S. The Mannose Receptor: From Endocytic Receptor and Biomarker to Regulator of (Meta)Inflammation. Front Immunol. 2021;12:765034.
Liu YT, Sun ZJ. Turning cold tumors into hot tumors by improving T-cell infiltration. Theranostics. 2021;11:5365-5386.
Langle YV, Balarino NP, Belgorosky D, Cresta Morgado PD, Sandes EO, Marino L, Bilbao ER, Zambrano M, Lodillinsky C, Eiján AM. Effect of nitric oxide inhibition in Bacillus Calmette-Guerin bladder cancer treatment. Nitric Oxide. 2020;98:50-59.
Bruchard M, Geindreau M, Perrichet A, Truntzer C, Ballot E, Boidot R, Racoeur C, Barsac E, Chalmin F, Hibos C, et al. Recruitment and activation of type 3 innate lymphoid cells promote antitumor immune responses. Nat Immunol. 2022;23:262-274.
Mathew AA, Zakkariya ZT, Ashokan A, Manohar M, Keechilat P, Nair SV, Koyakutty M. 5-FU mediated depletion of myeloid suppressor cells enhances T-cell infiltration and anti-tumor response in immunotherapy-resistant lung tumor. Int Immunopharmacol. 2023;120:110129.
Galon J, Bruni D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. Nat Rev Drug Discov. 2019;18:197-218.
Grasselly C, Denis M, Bourguignon A, Talhi N, Mathe D, Tourette A, Serre L, Jordheim LP, Matera EL, Dumontet C. The Antitumor Activity of Combinations of Cytotoxic Chemotherapy and Immune Checkpoint Inhibitors Is Model-Dependent. Front Immunol. 2018;9:2100.
Sleijfer S, Bannink M, Van Gool AR, Kruit WH, Stoter G. Side effects of interferon-alpha therapy. Pharm World Sci. 2005;27:423-431.
Trevaskis NL, Kaminskas LM, Porter CJ. From sewer to saviour - targeting the lymphatic system to promote drug exposure and activity. Nat Rev Drug Discov. 2015; 14:781-803.
Trubetskoy VS, Whiteman KR, Torchilin VP, Wolf GL. Massage-induced release of subcutaneously injected liposome-encapsulated drugs to the blood. J Control Release. 1998;50:13-19.
Bocci V, Pessina GP, Paulesu L, Nicoletti C. The lymphatic route. VI. Distribution of recombinant interferon-alpha 2 in rabbit and pig plasma and lymph. J Biol Response Mod. 1988;7:390-400.
MacNabb BW, Tumuluru S, Chen X, Godfrey J, Kasal DN, Yu J, Jongsma MLM, Spaapen RM, Kline DE, Kline J. Dendritic cells can prime anti-tumor CD8(+) T cell responses through major histocompatibility complex cross-dressing. Immunity. 2022; 55:982-997.e988.
Gray EE, Cyster JG. Lymph node macrophages. J Innate Immun. 2012;4:424-436.
Zhu M. Immunological perspectives on spatial and temporal vaccine delivery. Adv Drug Deliv Rev. 2021;178:113966.
Pilkington EH, Suys EJA, Trevaskis NL, Wheatley AK, Zukancic D, Algarni A, Al-Wassiti H, Davis TP, Pouton CW, Kent SJ, Truong NP. From influenza to COVID-19: Lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases. Acta Biomater. 2021;131:16-40.
Sakumura N, Yokoyama T, Usami M, Hosono Y, Inoue N, Matsuda Y, Tasaki Y, Wada T. CD169 expression on monocytes as a marker for assessing type I interferon status in pediatric inflammatory diseases. Clin Immunol. 2023;250:109329.
Minayoshi Y, Maeda H, Yanagisawa H, Hamasaki K, Mizuta Y, Nishida K, Kinoshita R, Enoki Y, Imafuku T, Chuang VTG, et al. Development of Kupffer cell targeting type-I interferon for the treatment of hepatitis via inducing anti-inflammatory and immunomodulatory actions. Drug Deliv. 2018;25:1067-1077.

No competing interests reported.

Download PDF

Editorial decision: Revision requested
26 Sep, 2024
Reviews received at journal
10 Sep, 2024
Reviews received at journal
09 Sep, 2024
Reviews received at journal
04 Sep, 2024
Reviewers agreed at journal
04 Sep, 2024
Reviewers agreed at journal
30 Aug, 2024
Reviewers agreed at journal
30 Aug, 2024
Reviewers agreed at journal
30 Aug, 2024
Reviewers invited by journal
30 Aug, 2024
Editor assigned by journal
21 Aug, 2024
Submission checks completed at journal
21 Aug, 2024
First submitted to journal
20 Aug, 2024

You are reading this latest preprint version

Lymph node macrophage-targeted interferon alpha boosts anticancer immune responses by regulating CD169-positive phenotype of macrophages

Status:

Version 1

Abstract

Figures

Background

Methods

Results

Spatial identification of LN cells in cancer patients

Lymphatic drainage of cancer antigens in tumor-bearing mice

Expression of mannose receptors and type Ⅰ IFN receptors in LN macrophages

Lymphatic drainage of albumin in mice

Design and physicochemical properties of Man-MSA-mIFNα

Intralymph node distribution of Man-MSA-mIFNα and its capacity to induce the CD169⁺ phenotype of macrophages

Antitumor effect of Man-MSA-mIFNα in tumor-bearing mice

Antitumor effect of Man-MSA-mIFNα in CD169⁺ macrophage-depleted mice

Antitumor effect of a combination therapy of Man-MSA-mIFNα and PD-L1 blockade

Safety evaluation

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Lymph node macrophage-targeted interferon alpha boosts anticancer immune responses by regulating CD169-positive phenotype of macrophages

Status:

Version 1

Abstract

Figures

Background

Methods

Results

Spatial identification of LN cells in cancer patients

Lymphatic drainage of cancer antigens in tumor-bearing mice

Expression of mannose receptors and type Ⅰ IFN receptors in LN macrophages

Lymphatic drainage of albumin in mice

Design and physicochemical properties of Man-MSA-mIFNα

Intralymph node distribution of Man-MSA-mIFNα and its capacity to induce the CD169+ phenotype of macrophages

Antitumor effect of Man-MSA-mIFNα in tumor-bearing mice

Antitumor effect of Man-MSA-mIFNα in CD169+ macrophage-depleted mice

Antitumor effect of a combination therapy of Man-MSA-mIFNα and PD-L1 blockade

Safety evaluation

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Intralymph node distribution of Man-MSA-mIFNα and its capacity to induce the CD169⁺ phenotype of macrophages

Antitumor effect of Man-MSA-mIFNα in CD169⁺ macrophage-depleted mice