Cell and fluid sampling microneedle patches for monitoring skin-resident immunity

Abstract

Important cell populations reside within tissues and are not accessed by traditional blood draws used to monitor the immune system. To address this issue at an essential barrier tissue, the skin, we created a microneedle-based technology for longitudinal sampling of cells and interstitial fluid, enabling minimally invasive parallel monitoring of immune responses. Solid microneedle projections were coated by a cross-linked biocompatible polymer, which swells upon skin insertion, forming a porous matrix for local leukocyte infiltration. By embedding molecular adjuvants and specific antigens encapsulated in nanocapsules within the hydrogel coating, antigen-specific lymphocytes can be enriched in the recovered cell population, allowing for subsequent detailed phenotypic and functional analysis. We demonstrate this approach in mice immunized with a model protein antigen or infected in the skin with vaccinia virus. After vaccination or infection, sampling microneedles allowed tissue-resident memory T cells (T_RMs) to be longitudinally monitored in the skin for many months, during which time the antigen-specific T cell population in systemic circulation contracted to low or undetectable counts. Sampling microneedles did not change the immune status of naïve or antigen-exposed animals. We also validated the ability of cell sampling using human skin samples. This approach may be useful in vaccines and immunotherapies to temporally query T_RM populations or as a diagnostic platform to sample for biomarkers in chronic inflammatory and autoimmune disorders, allowing information previously accessible only via invasive biopsies to be obtained in a minimally invasive manner from the skin or other mucosal tissues.

Figure 1:. Fabrication of Stimulatory Sampling Microneedles (SSMNs) platform for immune monitoring.

(A) Schematic of SSMNs structure and proposed mechanisms of action. (B) Microneedle fabrication process. (C) Trypan blue stain of mouse ear tissue. Scale bar 500 μm. (D-F) Confocal micrographs showing a cross section of the alginate layer (blue) on an individual microneedle projection before (D) and after (E) swelling in PBS for 20 minutes at 25°C (scale bar 50 μm). (F) Thickness of alginate layer quantified before/after PBS swelling.

Figure 2:. Cell sampling microneedles allow tandem analysis of cellular and humoral immune responses.

(A) Groups of OVA-immunized or naïve C57Bl/6 mice (n=9/group) were injected intradermally in the ear at time zero with 2 μg OVA and 5 μg each of adjuvants polyI:C and pam3Cys. 60 hrs later, sampling microneedle were applied to the same site for 12 hrs, followed by retrieval and flow cytometry analysis. (B) Sample processing and flow cytometry gating strategy. (C-D) Representative flow cytometry plots (C) and quantification from groups of animals (D) showing OVA-specific SIINFEKL/H-2K^b-streptavidin tetramer⁺ CD8⁺ cells, as sampled from blood or with cell-sampling microneedles. (E) OVA-specific IgG titers(log10) as quantified from serum or ISF from sampling microneedles. Data shown are means ± s.e.m. from one representative of two independent experiments. ns, nonsignificant, **, p < 0.01, ****, p < 0.0001, analyzed by one-way ANOVA, followed by Tukey’s HSD.

Figure 3:. Microneedles incorporating adjuvants enable single-step sampling of antigen-specific T_RMs.

(A) Timeline of sampling optimization in C57Bl/6 mice (n=5/group). (B-G): Enumeration of recovered total live cells (B), CD8⁺ cells (C), OVA-specific CD8⁺ cells (D), frequency of OVA-specific cells (E), OVA-specific T_RMs (F), and frequency of T_RMs among OVA-specific cells (G). Cell counts are per microneedle patch. Data shown are mean ± s.e.m. from one representative of three independent experiments. *, p<0.05, **, p<0.01, and ****, p < 0.0001 analyzed by one-way ANOVA, followed by Tukey’s HSD.

Figure 4:. SSMNs containing antigen-loaded nanocapsules enrich sampling for antigen-specific T_RMs.

(A-E) Groups of OVA-immunized C57Bl/6 mice (n=6/group) were sampled with SSMN (Stimulatory Sampling Microneedle) arrays applied to the ear according to the timeline (A). Enumeration of recovered total live cells (B), CD8⁺ cells (C), CD8⁺CD69⁺CD103⁺ T_RMs (D) and OVA-specific T_RMs (E), per SSMN array, from SSMNs containing empty ICMV nanocapsules or ICMVs loaded with 2 μg OVA. (F-J) Groups of OVA-immunized C57Bl/6 mice (n=5/group) were sampled with microneedles applied to the ear according to the timeline (F). Enumeration of recovered total live cells (G), CD8⁺ cells (H), CD8⁺CD69⁺CD103⁺ T_RMs (I) and OVA-specific T_RMs (J), per SSMN array, from SSMNs containing ICMVs encapsulating 0.02 μg, 0.2 μg or 2 μg OVA. Data shown are mean ± s.e.m. from one representative of 2-3 independent experiments. **, p<0.01, *** and p < 0.001 analyzed by one-way ANOVA, followed by Tukey’s HSD.

Figure 5:. Sampling microneedles containing ICMV nanocapsules accumulate antigen-loaded dendritic cells.

(A) Confocal micrograph of SSMN array surface showing alginate (blue), embedded ICMVs (red), and recruited cells (green), following application to the skin of eGFP mice. Scale bar 20 μm. (B) SEM of SSMNs post-application to mouse skin for 24 hours showing lymphocytes, approximately 5μm in diameter, (C). (D-G) SSMNs containing 5 μg of polyI:C and DiD-labeled ICMVs encapsulating 2 μg Alexa Fluor555-labeled OVA and 5 μg pam3Cys were applied to the ears of OVA-immunized mice (n=3/group) for 24 hours, followed by retrieval, antibody staining and phenotypic analysis via imaging cytometry (D). (E) Cell phenotypes obtained from SSMNs. (F) Representative imaging cytometry data showing overlay of fluorescent channels for live/dead dye Sytox, CD45, CD3, CD19, CD11c, DiD (ICMV) and OVA-Alexa Fluor555. Dimension of each well is 50 μm. (G) Representative 2D imaging cytometry plots showing OVA/ICMV fluorescence in CD45⁺CD11c⁺ dendritic cells. (H-I) Expression of activation markers CD40 (H) and CD86 (I) among APCs recovered by microneedles. Data shown are mean ± s.e.m. from one representative of 2-3 independent experiments. *, p<0.05, **, and p<0.01 analyzed by one-way ANOVA, followed by Tukey’s HSD.

Figure 6:. Sampling microneedles do not change the immune status of the animal.

SSMN arrays containing adjuvants and ICMVs loaded with 2 μg of OVA were applied to the ears of naïve or OVA-immunized C57Bl/6 mice, immunized 8-10 weeks prior, (n=5/group) for 24 hrs, then retrieved and analyzed via flow cytometry. Shown are experimental timeline (A), representative flow cytometry plots and quantification of OVA-specific CD8⁺ T cells from blood in naïve (B) and previously immunized (C) mice, before and after SSMN application at day 0 and vaccination on day 24. (D) Serum OVA-specific IgG titers pre- and post-sampling with SSMNs. Data shown are mean ± s.e.m. from one representative of two independent experiments. ****: p < 0.0001 analyzed by one-way ANOVA, followed by Tukey’s HSD.

Figure 7:. Sampling microneedles reveal a stable population of T_RMs in skin up to a year post vaccination or infection.

(A-D) Groups of C57Bl/6 mice (n=5/group) were primed and boosted with OVA and adjuvant, and then repeatedly sampled using SSMN arrays and blood draws over time. (A) Timeline. (B) OVA-specific CD8⁺ T cells over time. (C-D), T_RMs (C) and OVA-specific T_RMs (D), quantified from SSMNs and blood. (E-H) Groups of C57Bl/6 mice (n=5/group) were infected with 2x10⁶ PFU of SIVgag-expressing vaccinia virus via tail skin scarification and sampled beginning 11 weeks post infection via blood draws or SSMNs applied to the ear containing ICMVs (2 μg AL11 SIVgag peptide and 5 μg pam3Cys) and 5 μg polyI:C. After 24 hrs, patches were retrieved and analyzed via flow cytometry. Experimental timeline (E), frequency of SIVgag tetramer⁺CD8⁺ cells (F), enumeration of CD8⁺CD69⁺CD103⁺ T_RM cells (G) and SIVgag-specific T_RMs (H) from blood and SSMNs. (I-K) Timeline (I), frequency of SIVgag-specific CD8⁺ T cells (J) and frequency of SIVgag-specific T_RMs (K), when sampled at various times post vaccinia infection. Data shown are mean ± s.e.m. from one representative of 2-3 independent experiments, analyzed by Wilcox-Mann-Whitney test (F-H), one-way ANOVA (K) or two-way ANOVA (B, J), ns, nonsignificant, **p < 0.01, ***p < 0.001, ****, p < 0.0001.

Figure 8:. SSMNs enable sampling of lymphocytes from human skin.

Five SSMN arrays each were applied to excised human skin samples from n=5 donors for 16 hrs, and then gel coatings were digested for analysis of recovered cells via flow cytometry. (A) Timeline and photographs of the experimental setup. (B-D) Enumeration of recovered total live cells (B), CD8⁺ cells (C), CD11c⁺CD45⁺ cells (D), per sampling microneedle array, from microneedles containing no adjuvants, or SSMNs containing polyI:C and ICMVs encapsulating pam3Cys. Data shown are mean ± s.e.m. from two independent experiments with samples obtained from 5 total donors, analyzed by two-tailed nonparametric Mann-Whitney test, **p < 0.01.