Coformulation with Tattoo Ink for Immunological Assessment of Vaccine Immunogenicity in the Draining Lymph Node

Peripheral lymphoid tissues, including lymph nodes (LN), tonsils, and mucosal-associated lymphoid tissues, are critical sites for the generation of adaptive immunity and immunological memory. After i.m. administration, vaccine Ags drain via the lymphatics to be concentrated and retained within the LN, where they are subject to immune surveillance. Abs are a key protective correlate for most human vaccines, with high-affinity variants generated within germinal centers (GC) via tightly regulated interactions of Ag, GC B (B_GC) cells, and T follicular helper (T_fh) cells (1). Efficient generation of GCs by immunization is therefore a key determinant of vaccine success or failure, controlling the kinetics, magnitude, and quality of the resultant serological response (2, 3). Direct characterization of Ag-specific B_GC or T_fh cells can provide important insights into vaccine immunogenicity and the biogenesis of protective immune responses. However, interrogation of LN B_GC and T_fh cells in humans is challenging, requiring invasive surgical excision or the collection of a small number of cells by fine needle aspirates (4, 5). In contrast, preclinical animal models such as nonhuman primates (NHPs) offer the opportunity to collect longitudinal LN and peripheral blood samples during vaccine studies (6, 7). A factor critical to the detection of these immune responses is the accurate sampling of LN that drains the injection site, which can be technically challenging because of the sporadic route of Ag trafficking in vivo.

There are multiple factors that can confound accurate sampling of vaccine-draining LNs. In humans, i.m. vaccination into the deltoid muscle sees Ags drain predominately to the axillary LNs, with vaccine responses preferentially draining to LNs in anatomic proximity to the injection site (8–12). Vaccination in the quadriceps femoris muscle group is expected to drain predominately to the deep inguinal LN, which subsequently drains to the external iliac LN in the pelvis (9, 13–15). In some individuals, lymphatic drainage from the thigh musculature may bypass the ipsilateral inguinal LNs and drain directly into the iliac LNs (14–16). Whereas lymphatic drainage patterns of the thoracic limb are conserved between rodents, NHPs, and humans (2, 17, 18), pelvic limb lymphatics predominately drain to the iliac LNs in mice and NHPs, with inconsistent drainage to the ipsilateral inguinal LN (2, 18, 19). In larger species, LNs are grouped in clusters within individual anatomic sites with dissimilar amounts of Ag deposition, further complicating accurate sampling of the vaccine-draining LN (2, 18, 20, 21). We observed substantial variability in vaccine-induced responses when random LNs in the draining region are sampled in NHPs (20), likely in part because of not sampling the particular responding LN among a cluster. The ability to directly track Ag drainage following vaccination would substantially improve the accuracy of serial LN biopsies and assessment of GC immune responses, particularly in large animals such as NHPs. Although this can be partially mitigated by substituting s.c. for i.m. vaccine administration (2), the majority of human vaccines are given i.m., and it is desirable to maintain comparable delivery in preclinical animal models.

Previous studies have used tracking dyes to broadly identify LN drainage patterns in rodent and NHP animal models (2, 17–19, 21–23). Tracking dyes have also been used clinically to identify sentinel LNs in cancer patients for biopsy or surgical resection (24–28). However, the potential for mixing vaccine Ags with tracking dyes for long-term demarcation of draining LNs in vivo is untested. In this study, we show that that coformulating influenza hemagglutinin (HA) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike immunogens with adjuvant and tattoo ink allows for the ready visual identification of draining LNs in mice and NHPs without compromising downstream analyses of cellular and humoral immunity. We propose tattoo ink–based vaccine tracking as an effective method for the differentiation of vaccine-draining from nondraining LNs during extended periods of time postvaccination. This technique facilitates a more accurate quantification and phenotypic characterization of vaccine-specific B_GC and T_fh cells in draining LNs in both murine and NHP animal models.

Mouse studies and related experimental procedures were approved by the University of Melbourne Animal Ethics Committee (identification no. 1914874). Female C57BL/6JArc mice (6–8 wk old) were anesthetized by isoflurane inhalation prior to immunization. For i.m. vaccinations, 5 μg of PR8-HA protein with AddaVax (1:1 ratio; InvivoGen) or 2% Alhydrogel (1:1 ratio; InvivoGen) and 0.5% tattoo ink (total volume: 50 μL per site) was injected in the quadriceps femoris or gastrocnemius using a 30-G needle. At the reported time points postvaccination, the mice were euthanized via CO₂ asphyxiation, and relevant draining LNs (inguinal, iliac, and ^+/−popliteal) and nondraining LNs (axillary) were collected for further analysis.

Macaque studies and related experimental procedures were approved by the Monash University Animal Ethics Committee (identification no. 23997). Pigtail macaques (Macaca nemestrina) were housed in the Monash Animal Research Platform Gippsland Field Station. Eight male pigtail macaques (M. nemestrina) (6–15 y old) were initially vaccinated with 100 μg of SARS-CoV-2 spike protein (S), consisting of either the whole S or the receptor binding domain of the S (RBD), with 200 μg of monophosphoryl lipid A (MPLA) adjuvant i.m. in the right quadriceps femoris (total volume: 1 ml). A booster vaccine of 100 μg S or RBD protein with 200 μg of MPLA and 1% tattoo ink was administered i.m. in both quadriceps femoris muscles (total volume: 500 μL per site) 28 d after the initial vaccine. The macaques were concurrently vaccinated in the right and left deltoid muscles with HIV-1 fixed trimeric envelope protein gp140 vaccines (100 μg) (29, 30) formulated with MPLA (100 μg) and 1.0% tattoo ink (right deltoid only) (total volume: 1 ml per site). Twenty-four hours prior to necropsy, the macaques received an i.v. infusion of autologous Vδ2⁺Vγ9⁺ T cells labeled with CellTrace Blue (Life Technologies). Thirteen to fourteen days post–booster vaccination, the macaques were euthanized by barbiturate overdose, and draining and nondraining LNs were collected for further analysis. Detailed immunogenicity studies of the vaccines used will be reported separately to this publication.

Fresh or cryopreserved human PBMCs were isolated from heparinized whole blood by Ficoll gradient (Sigma). Cells were incubated for 1 hr (37°C and 5% CO₂) in RPMI-1640 (Life Technologies) supplemented with 10% FCS and 5% penicillin/streptomycin/glutamine. Samples were incubated with 0.05% w/v Evans blue dye (Sigma Life Sciences), 1.0% v/v India ink (Higgins waterproof ink), or 1.0% v/v tattoo ink (Moms Millennium Black Pearl) in six-well plates at ∼1.5–2.0 × 10⁶ cells/ml. Control samples were incubated in media without dye under identical conditions. Samples were washed two times with PBS prior to analysis.

Full-length influenza H1N1 A/Pu (PR8-HA) (20, 31), SARS-CoV-2 S (32), and the RBD (32) were prepared as previously described and used for immunogens, flow cytometric assays, and serological assays. PR8-HA and SARS-CoV-2 S proteins with C-terminal Avi-tags and His-tags were expressed via transient transfection of Expi293 suspension cultures (Thermo Fisher Scientific) and purified by polyhistidine-tag affinity and size exclusion chromatography. For flow cytometry B cell probes, purified PR8-HA and S proteins were biotinylated using BirA biotin–protein ligase (Avidity Biosciences). Biotinylated PR8-HA and S proteins were fluorescently labeled by the sequential addition of streptavidin-conjugated PE (Thermo Fisher Scientific) prior to use.

Murine LNs were processed and analyzed individually. Prior to processing, all LNs were photographed and noted for the presence or absence of visible tattoo ink. NHP LNs from each animal and anatomical location were separated based on the visual presence or absence of ink and processed as a pooled sample. Murine and NHP LNs were dissociated and passed through a 70-μm filter to generate single-cell suspensions. Murine LN suspensions were freshly stained for Ag-specific B cells. NHP LN suspensions were cryopreserved in 90% FCS/10% DMSO.

The amount of tattoo ink deposition in murine draining LNs was estimated based on the amount of visible tattoo ink on the surface of the LN. Vaccine-draining LNs (right and left inguinal LNs, right and left iliac LNs, and ^+/−right popliteal LN) and nondraining LNs (left popliteal LN and right and left axillary LNs) were evaluated 14 d post–i.m. vaccination with PR8-HA (5 μg) protein with AddaVax and tattoo ink. Draining and nondraining LNs were photographed for analysis, and the images were analyzed using FIJI/ImageJ (33, 34). The photographs were converted to 8-bit images, and individual LNs were outlined with the polygonal region of interest tool. The amount of tattoo ink deposition in each individual LN was then estimated by measuring the median intensity value within each region of interest. The median values were subsequently plotted on an 8-bit grayscale (0–255), in which lower median values correspond to darker LNs, which indicates more tattoo ink uptake.

Fresh, single-cell suspensions of individual murine LN were stained with LIVE/DEAD Aqua Viability Dye (Life Technologies) and Fc blocked with CD16/CD32 (93, BioLegend). Surface staining was then performed with the following Abs: GL7 Alexa Fluor 488 (GL7, BioLegend), CD45 allophycocyanin-Cy7 (30-F11, Becton Dickinson [BD]), F4/80 Brilliant Violet (BV) 786 (BM8, BioLegend), CD3ε BV786 (145-2C11, BioLegend), CD38 PE-Cy7 (90, BioLegend), IgD BUV395 (11-26c.2a, BD), and B220 BUV737 (RA3-6B2, BD). Biotinylated PR8-HA with streptavidin–PE (Thermo Fisher Scientific) was used to identify PR8-HA–specific B_GC cells, and streptavidin-BV786 (BD) was included to exclude nonspecific streptavidin binding to the cell surface. After 30-min incubation at 4°C, cells were washed and fixed in BD Cytofix Fixation Buffer. Samples were acquired on a BD LSRFortessa using BD FACSDiva.

For NHP B cell analysis, thawed LN single-cell suspensions were stained with LIVE/DEAD Aqua Viability Dye (Life Technologies) and the following surface Abs: IgD Alexa Fluor 488 (polyclonal, SouthernBiotech), CD20 allophycocyanin-Cy7 (2H7, BioLegend), CD14 BV510 (M5E2, BioLegend), CD3 Alexa Fluor 700 (SP34-2, BD), CD8α BV510 (RPA-T8, BioLegend), CD16 BV510 (3G8, BioLegend), CD10 BV510 (HI10a, BioLegend), IgG BV786 (G18-145, BD), CD95 BUV737 (DX2, BD), CD4 BV605 (L200, BD), CXCR5 PE-Cy7 (MU5UBEE, eBioscience), and PD-1 BV421 (EH12.2H7, BioLegend). S-specific B cells were identified using biotinylated SARS-CoV-2 S probe conjugated to streptavidin–PE (Thermo Fisher Scientific). Streptavidin-BV510 (BD) was included to exclude nonspecific streptavidin binding to the cell surface. Cells were washed and permeabilized with Transcription Factor Buffer Set (BD) prior to BCL-6 Alexa Fluor 647 (IG191E/A8, BioLegend) and Ki-67 BUV395 (B56, BD) staining. Samples were acquired on a BD LSRFortessa using BD FACSDiva.

For T cell analysis, single-cell suspensions were stained with LIVE/DEAD Aqua Viability Dye and the following cell surface markers: CD20 BV510 (2H7, BD), CD3 Alexa Fluor 700 (SP34-2, BD), CD4 BV605 (L200, BD), CD8 (RPA-T8, BioLegend), CXCR5 PE (MU5UBEE, Thermo Fisher Scientific), PD-1 BV421 (EH12.2H7, BioLegend), CD95 BUV737 (DX2, BD), CD25 allophycocyanin (BC96, BioLegend), and OX-40 BUV395 (L106, BD). After 30-min incubation at 4°C, cells were washed and fixed in 1% formaldehyde. Samples were acquired on a BD LSRFortessa using BD FACSDiva.

Mouse serum Ab binding to PR8-HA was evaluated by ELISA as previously described (35, 36). The 96-well Nunc MaxiSorp plates (Thermo Fisher Scientific) were coated overnight with 2 µg/ml of PR8-HA at 4°C. Wells were blocked with 1% FCS in PBS and incubated with serially diluted serum for 2 h at room temperature. Plates were washed and incubated with HRP-conjugated anti-mouse IgG secondary Abs (1:15,000 dilution in PBS/1% FCS, KPL/SeraCare) for 1 h at room temperature. Plates were developed using tetramethylbenzidine liquid substrate (Sigma) and read at 450 nm. Serum titers were calculated as the reciprocal serum dilution giving signal 2× background using a fitted curve (four parameter log regression).

Data are presented as median and interquartile range. Spearman correlation was used to compare the frequencies of B_GC and PR8-HA–specific B_GC cells with the median gray value of the corresponding LN. All statistical analysis data presentation was performed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA). Flow cytometry data were analyzed in FlowJo v9 or v10.

Tracking dyes used in vaccinations should 1) visibly stain the draining LN with codeposition of Ag and 2) not affect vaccine immunogenicity or downstream analyses, such as flow cytometric quantification of Ag-specific B_GC and T_fh populations. We first tested the impact of three candidate tracking dyes (Evans blue dye, India ink, and tattoo ink) on cell viability and autofluorescence in vitro. Human PBMCs cultured in media with 0.05% Evans blue dye for 1 h resulted in substantial cytotoxicity and loss of lymphocytes (Fig. 1A). In contrast, culture with 1% India ink or 1% tattoo ink did not affect lymphocyte viability (Fig. 1B). Despite the short duration of coculture (1 hr), incubation of PBMCs with 1% India ink demonstrated alterations in cellular autofluorescence as measured by flow cytometry on channels off the blue, violet, and UV lasers (Fig. 1C). Given tattoo ink demonstrated less autofluorescence (Fig. 1C), we proceeded to test the utility of tattoo ink coformulated with vaccine Ags in mice.

C57BL/6J mice were immunized i.m. in the right gastrocnemius and left quadriceps with influenza A/Puerto Rico/8/1934 HA (PR8-HA; 5 μg) formulated with AddaVax adjuvant and tattoo ink to evaluate the utility of tattoo ink as an in vivo tracking dye. In vivo titration of our tattoo ink coformulation showed robust ink deposition with 0.5% tattoo ink, with a progressive loss of visible tattoo ink with increasing dilutions (Supplemental Fig. 1). Based on these results, 0.5% tattoo ink was used for subsequent murine studies. Ags delivered to the right gastrocnemius will predominately drain to the right popliteal LN and the right iliac LN, with variable drainage to the right inguinal LN (Fig. 1D) (17, 19). Ags delivered in the left quadriceps will predominately drain to the left iliac LN, with variable drainage to the left inguinal LN (Fig. 1D). LN were harvested and assessed visually for ink staining 14 d postvaccination. Following i.m. vaccination in the left quadriceps, nondraining LN (left popliteal and axillary LN) showed no evidence of ink uptake, with the left iliac LN consistently exhibiting ink uptake (Fig. 1E, Table I). In contrast, right popliteal and right iliac LNs exhibited obvious ink uptake by eye following i.m. vaccination in the right gastrocnemius, whereas it is absent in the right axillary LN (Fig. 1E, Table I). These observations are consistent with previous reports that lymphatics from the pelvic limbs drain into the iliac LNs in mice (17, 19). Dye labeling of inguinal LN was variable, with ∼14% of the left inguinal LNs and 43% of the right inguinal LNs labeled (Fig. 1E, Table I). This may reflect differences in lymphatic drainage patterns between proximal and distal muscle groups of the pelvic limb.

To evaluate the stability of the tattoo ink staining of vaccine-draining LNs beyond 14 d, C57BL/6J mice were immunized i.m. in the right quadriceps with PR8-HA (5μg) with AddaVax and tattoo ink (0.5%) and serially sacrificed at 4, 6, and 8 wk postimmunization. Tattoo ink was readily identified in the right inguinal and iliac LNs at all time points following immunization (Fig. 1F, Table II). No ink uptake was observed in the left inguinal, iliac, or axillary LN, indicating that ink drainage is restricted to vaccine-draining LN. Overall, our results indicate that tattoo ink can label draining LNs of mice when administered in combination with Ag and will persist in draining LNs for extended periods of time.

To assess the extent to which ink staining tracks with vaccine-induced GC activity in mice, we quantified both total B_GC (B220⁺IgD^-GL7⁺CD38^dim) or HA-specific B_GC cell frequency (36) in LNs with and without gross ink uptake (Fig. 2A; gating in Supplemental Fig. 2A). Expanded frequencies of B_GC cells were observed in ink-dyed draining LN compared with nondraining LN or undyed draining LN (median 24.6% for ink-dyed LN versus 4.8% for nondraining LN and 8.9% for undyed draining LN; (Fig. 2B). Similarly, HA-specific B_GC cells were substantially enriched in draining ink-dyed LN (median 10.2% of total B_GC) compared with nondraining (median 0.0% of total B_GC) or undyed draining LN (median 1.1% of total B_GC) (Fig. 2B). These data indicate that the tattoo ink vaccine formulation does not hinder the identification of total or Ag-specific B_GC by flow cytometry and that ink-dyed draining LN are more likely to contain vaccine responses in the acute postvaccination period compared with undyed LN. Similar results were obtained with HA coformulated with Alhydrogel (alum) and tattoo ink (Supplemental Fig. 2B). To confirm that coformulation with tattoo ink does not adversely impact vaccine immunogenicity, we vaccinated mice in the quadriceps with either HA/AddaVax or HA/AddaVax/tattoo ink and collected all inguinal and iliac LN at day 14 postvaccination. Serological HA IgG titers were comparable between both groups as were the frequencies of B_GC or HA-specific B_GC in the draining LN (Fig. 2C), suggesting that tattoo ink does not modulate vaccine immunogenicity.

To further evaluate the utility of tattoo ink in labeling vaccine-draining LNs, we performed a quantitative evaluation of the amount of tattoo ink on the surface of draining and nondraining LNs 2 wk postvaccination and evaluated the correlation to the frequency of B_GC cells and HA-specific B_GC cells. Quantitation of tattoo ink deposition in LNs was accomplished by identifying the median intensity value of the surface of the LN, which were taken from gross LN images that were converted from RGB to 8-bit images. The median values were subsequently plotted on 8-bit grayscale (0–255), in which lower median values correspond to darker LNs, which indicates more tattoo ink uptake. Draining LNs with visible tattoo ink clustered toward lower median gray values, whereas nondraining LNs and draining LNs without gross tattoo ink deposition clustered toward higher median gray values, indicating that median gray values are a suitable quantitative approximate of tattoo ink deposition in LNs. Furthermore, median gray values in murine draining LNs decrease with increasing tattoo ink concentrations, which is not observed in nondraining LNs (Supplemental Fig. 3). This supports that median gray value intensity is an accurate metric to determine tattoo ink deposition in murine LNs. Draining LN median gray values significantly negatively correlated with both total and HA-specific B_GC (p = 0.049 and 0.023, respectively; (Fig. 2D) in mice immunized with PR8-HA coformulated with tattoo ink, suggesting that the degree of ink accumulation mirrors Ag load in the LN following vaccination.

Whereas mice generally have only 1–2 LNs at each lymphoid site (17, 19), primates and humans commonly exhibit chains or clusters of 2–25 LNs (9, 13, 15, 18, 21). This creates additional complexities when evaluating the adaptive immune response as vaccine Ag may drain to a small subset of the LNs at a given site. We tested if sampling accuracy, and the characterization of vaccine-elicited immune responses ex vivo, could be improved using tattoo ink to label vaccine-draining LNs in NHPs.

Pigtail macaques (M. nemestrina) were immunized in the right quadriceps with SARS-CoV-2 S (100 μg) formulated with MPLA liposomal adjuvant (37) and were boosted i.m. in the right and left quadriceps with SARS-CoV-2 S (100 μg) formulated with MPLA and tattoo ink (1.0%). Administration at these sites was expected to drain primarily to the left and right iliac LNs, with inconsistent drainage to the left and right inguinal LNs (Fig. 3A) (2, 18). Animals were additionally immunized in the right and left deltoids with HIV-1 fixed trimeric envelope protein gp140 vaccines (100 μg) formulated with MPLA and 1.0% tattoo ink (ink in the right deltoid only), with expected drainage to the ipsilateral axillary LNs (Fig. 3A) (2, 18). Animals were humanely euthanized 13–14 d after the second immunization, and necropsies were performed to evaluate tattoo ink deposition in draining and nondraining LNs. Among the draining LNs, tattoo ink was visible in at least one LN within the right and left iliac chains in seven of eight animals at necropsy (Table III). Dye labeling of inguinal LNs was variable, with LNs containing tattoo ink being identified in the left and right inguinal lymphoid sites in one of eight and four of eight animals, respectively (Fig. 3B, 3C, Table III). Stochastic drainage to the ipsilateral inguinal LN aligns with previous reports (2) and is important to consider as the inguinal LN is a common and readily accessible site for LN sampling via fine needle aspirates or biopsy. Dye labeling was observed in at least one right axillary LN in eight of eight animals, whereas dye labeling in the left axillary LNs was observed in one LN within a cluster in one of eight animals (Fig. 3D–F, Table III). No tattoo ink was grossly visible in any nondraining LN clusters, including the popliteal, para-aortic, mesenteric, mediastinal, tracheobronchial, and submandibular LNs (data not shown). Tattoo ink was generally observed in only a single or limited number of LNs recovered from a given lymphoid site (Fig. 3B–F). This suggests that 1) randomly sampling a single LN within a cluster has a significant risk of missing the relevant draining node, and 2) pooling all LNs within a cluster for immunological analysis could result in significant dilution of vaccine-specific responses and unpredictable effects on reported frequencies of Ag-specific B and T cell responses.

Longitudinal studies involving LN biopsies in macaques commonly sample the more readily accessible inguinal LN, to which Ag drainage is highly stochastic (2). Among the eight animals vaccinated i.m. into the quadriceps, only four exhibited some degree of tattoo ink staining of inguinal LNs (NM11, NM88, NM224, and NM251). Samples from three animals (NM11, NM88, and NM224) were available to evaluate Ag-specific B_GC and T_fh frequencies in tattoo ink–containing LNs. Comparison of GC T_fh and SARS-CoV-2 S-specific B_GC populations among ink-dyed or undyed inguinal LN, as well as a nondraining LN control (submandibular), in NM11 showed a strong enrichment of vaccine responses and GC activity in the ink-stained nodes (Fig. 4A). In LN samples from NM88, only low levels of S-specific B cells were observed along with comparable GC T_fh frequencies among both draining (inguinal) and nondraining LN, suggesting that vaccine responses in this animal were restricted to the iliac LN (Fig. 4B). NM224 was the only animal without ink staining in the iliac LN, with dye accumulation only occurring in the inguinal LN. A comparison of the iliac and inguinal LN confirmed that, similar to NM11, vaccine-induced GC responses were associated with the ink-stained inguinal LN (Fig. 4C). Overall, these results demonstrate the utility of tattoo ink for ex vivo identification of draining LN enriched for vaccine-induced GC responses.

Draining LNs are key sites for the induction of adaptive immune responses following immunization and drive the development of immunological memory. A detailed understanding of complicated LN processes, such as the biogenesis of GCs and the induction of Ag-specific T and B cell responses, is key for rational design of next-generation vaccines, particularly for pathogens that have eluded traditional vaccine development efforts like HIV and malaria. Techniques enabling the targeted study of vaccine-draining LN allow for precise analyses of immunity without the confounding inclusion of nondraining LN.

In this study, we show that tattoo ink is an effective and durable tracking dye that can be formulated into vaccines without compromising flow cytometry–based analysis of B_GC and T_fh cells. Preliminary analysis also suggests that infiltration and activation of innate immune cells, including dendritic cells and macrophages, into the draining LN can be easily detected using tattoo ink staining (not shown). Deposited tattoo ink in LNs was associated with higher frequencies of immunogen-specific B_GC cells in mice and T_fh and immunogen-specific B_GC cells in NHPs. We observed high levels of specificity in vivo, with minimal staining observed in nondraining LN at distal sites and good concordance between staining and immunity within a single site. Importantly, the inclusion of tattoo ink in the vaccine formulation does not augment nor adversely impact on immunogenicity at the serological or cellular level. Precise sampling of vaccine-draining LNs, therefore, avoids the dilutional effects of sampling nondraining LNs from the same site and improves the accurate quantification of Ag-specific B and T cell responses in immunized animals.

Numerous studies employing a variety of different dyes have tracked lymphatic drainage in the hours or days after administration (2, 17–19, 21, 22, 24–28). Although studies have suggested that tracking dyes can rapidly travel through the lymphatic system to the LN via passive drainage (19), long-term stability in vivo and the influence of dye on downstream immunological analysis is unclear. Prolonged labeling of vaccine-draining LNs is a relevant consideration as it allows serial sampling to evaluate changes in the immune response over weeks to months after initial vaccination. Visible dye staining in rats was reported 10–14 d after i.p. administration of pontamine sky blue dye (23). Tattoo ink, by design, is a stable, relatively inert compound that can persist in LNs for extended periods of time. In humans, several case reports have noted tattoo ink being incidentally identified in draining LNs up to 30 y after the tattoo was originally placed (38–40). The longevity of tattoo ink in draining LNs when formulated with vaccines still needs to be determined. However, our data demonstrate that draining LNs containing tattoo ink can be readily identified 2 wk after administration in NHPs and 8 wk after administration in mice, with the likelihood of it persisting for considerably longer.

Published methods for identifying vaccine-draining LNs include administration of fluorescently labeled immunogens that can be identified by In Vivo Imaging Systems (2, 41) and administration of [⁹⁹Tc]sulfur colloid that can be identified with a γ probe (42). Vaccines may also be administered in specific anatomical locations or specific routes to increase the likelihood of Ag drainage to specific LNs, for example, injection in the s.c. flank in mice for selective drainage to the ipsilateral inguinal LN (43, 44) or s.c. immunization in the anterior thigh for selective drainage to the inguinal and iliac LNs in NHPs (2). The route of vaccine can impact the associated immune response, for example, reports of s.c. immunization eliciting stronger neutralizing Ab response compared with i.m. immunization in NHPs (45). Identification of vaccine-draining LNs with tattoo ink provides a simple approach for active LN identification without requiring specialized equipment while permitting i.m. vaccination routes of greatest clinical relevance for human vaccines.

One consideration with our proposed method is the potential for both endogenous and exogenous pigments to confound the identification of ink-containing LN. Hemosiderin, an iron-containing hemoglobin breakdown product, can accumulate in draining LN from congested, hemorrhagic, or inflamed areas (46–48). Similarly, carbon-containing, particulate debris can accumulate in tracheobronchial and mediastinal LN, following inhalation and drainage from the pulmonary tree (46, 47, 49). Accumulations of any of these pigments can cause LN darkening, which could be mistaken for tattoo ink accumulation and confound positive identification of relevant LN. Such hemosiderin accumulation in draining LN may have contributed to false identification of NM88 right inguinal LN and NM269 left axillary LN as tattoo ink–containing LNs in the current study. Tattoo ink comprising alternative colors could be explored to address this.

Overall, we find that coformulating immunogens with commercial tattoo ink does not hinder the adaptive immune response in the draining LN nor interfere with flow cytometry–based methods of immune analysis. Although the addition of tattoo ink did not impact the reported flow cytometry panels, it remains possible that other fluorophores may be negatively affected and compatibility with flow cytometry panels should be established in vitro prior to in vivo usage. Although this study focused on protein-based vaccines draining to LN in the pelvic limb, we show potential utility for fore-limb immunizations draining to the axillary LNs, and this approach could be extended to other tissues sites and other vaccine platforms. Increased accuracy in profiling vaccine immunity in key preclinical animal models is essential to guide the rational improvement of vaccines for human use.

The authors thank the staff at the Monash Animal Research Platform Gippsland Field Station, including Irwin Ryan, for assistance with the NHP study. We thank Robin Shattock (Imperial College London), Marit Van Gils (Amsterdam Medical Centre), and the European AIDS Vaccine Initiative consortium for the provision of HIV immunogens and Dietmar Katinger and Philipp Mundsperger from Polymun Scientific for provision of the MPLA liposome adjuvant.

The authors have no financial conflicts of interest.