IL-12 mRNA-LNP promotes dermal resident memory CD4+ T cell development

Dermal resident memory CD4+ T cells (dTrm) protect against vector-borne infections. However, the factors that promote their development remain unclear. We tested if an mRNA vaccine, encoding a protective leishmanial antigen, induced dTrm cells. The mRNA vaccine induced robust systemic T-cell responses, but few Trm cells were found in the skin. Since IL-12 promotes Th1 responses, we tested whether IL-12 mRNA combined with the mRNA vaccine could enhance dTrm cell development. This combination significantly expanded Leishmania-specific Th1 cells expressing skin-homing molecules and memory T cell markers in the draining lymph node. Additionally, higher numbers of dTrm cells were maintained in the skin, and mice exhibited functional immunity, indicated by a delayed hypersensitivity response and protection upon challenge with Leishmania. These findings highlight IL-12 as a key driver of CD4+ dTrm development, enabling their global seeding across the skin, and underscore the potential of IL-12-enhanced mRNA vaccines to generate durable immunity against cutaneous leishmaniasis and other skin-targeted infections.

Methods
Ethics statement

All animals were used in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the guidelines of the University of Pennsylvania Institutional Animal Use and Care Committee. The Institutional Animal Use and Care Committee approved the protocol.

Anesthesia/Euthanasia of mice

Mice were anesthetized by placing them in a plexiglass chamber with 4% isoflurane, USP (#NDC: 11695-6777-2, Covetrus, Dublin, OH, USA) for 4 min, or until fully sedated, as measured by lack of paw reflex, and then proceeded to any injection. When euthanized, mice were left in a Crainey Tech chamber with displacement of air with 100% CO2 for at least two minutes after respiration ceased. Then a cervical dislocation was performed to confirm death and proceed to tissue collection.

Mice and T cell transfer

Six-week-old female C57BL/6 and CD45.1 mice were purchased from Charles River and housed in the University of Pennsylvania Animal Care Facilities for 1–2 weeks before use. PEPCK-specific TCR transgenic mice (kindly provided by Dr Jude Uzonna) were bred in the University of Pennsylvania Animal Care Facilities. To create PEPCK chimera, spleen cells from PEPCK-specific TCR transgenic mice were collected, erythrocytes lysed with ACK lysing buffer (Quality Biological, Cat #118-156-101), and CD4+ T cells enriched using a magnetic bead separation kit (Miltenyi Biotec, Cat #130-104-075), 1 × 105 Ptg cells in 100 μL of dPBS were transferred by retroorbital injection into recipient mice.

Parasite culture and intradermal infection

L. major parasites (strain WHO/MHOM/IL/80/Friedlin) were grown in Schneider’s insect medium (GIBCO) supplemented with 20% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin per mL. Infective-stage promastigotes (metacyclics) were isolated from 4 to 5-day-old stationary culture by density gradient separation using Ficoll (Sigma-Aldrich, Cat #F9378). Mice were infected with 1 × 10^5 ^L. major metacyclics intradermally in the left ear. Lesion sizes were recorded weekly by measuring ear thickness with a digital caliper (Fisher Scientific). DTH was assessed by measuring ear thickness during the first 3 days of infection and compared with control infected mice that had not been immunized.

mRNA design and synthesis

The phosphoenolpyruvate carboxykinase (PEPCK) and IL-12 cytokine (IL-12) amino acid sequences underwent codon optimization and GC enrichment using our proprietary algorithm to improve expression and reduce potential immunogenicity of the in vitro transcribed mRNA. The codon optimized sequences were gene synthetized by Genscript, cloned into our proprietary in vitro transcription template containing an optimized T7 promoter, 3’UTR, 5’UTR and a 100-adenine tail. The PEPCK and IL-12 nucleoside modified mRNA sequences were prepared using the MegaScript transcription kit (ThermoFisher Scientific), co-transcriptionally capped using the 3’OMe CleanCap™ system (TriLink Biotechnologies) and purified using a modified cellulose-based chromatography, precipitated, eluted in nuclease free water, and quantified using the NanoDrop One system. Length and integrity were determined using the Agilent BioAnalyzer 2100 system. Endotoxin content was measured using the GenScript Toxisensor chromogenic assay, and values were below detection levels (0.1 EU/mL). mRNA was stored at −20 °C until formulation.

mRNA-LNP production and characterization

Purified mRNAs were formulated into lipid nanoparticles using a self-assembly process wherein an ethanolic lipid mixture of an ionizable cationic lipid, phosphatidylcholine, cholesterol, and polyethylene glycol-lipid was rapidly combined with an aqueous solution containing mRNA at acidic pH as previously described. The ionizable cationic lipid (pKa in the range of 6.0–6.5, proprietary to Acuitas Therapeutics) and LNP composition are described in the patent application WO 2017/004143. The hydrodynamic size, polydispersity index (PDI) and zeta potential of mRNA-LNPs were measured using a Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK). The mRNA encapsulation efficiency was determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen). Endotoxin levels were determined using the Limulus Amebocyte Lysate (LAL) chromogenic assay, found to be <0.5 endotoxin unit (EU)/mL.

Immunizations

All mice were immunized subcutaneously (s.c.) in their left footpad with the indicated dose of mRNA-LNP in 50 μL of PBS or 50 μL PBS alone as a control. In mice vaccinated with PEPCK mRNA LNP and IL-12 mRNA LNP, particles were mixed so that an equivalent LNP and antigen were given between groups. Peptide-immunized mice received PEPCK peptide (10 μg), CpG (50 μg), and rmIL-12 (0.5 μg) given in the footpad. Mice that received PEPCK T-cells were immunized 24 h later.

Tissue processing

To prepare a single-cell suspension of the skin samples, the dorsal and ventral layers of the ear were split while the footpad was cut into several pieces in a 24-well plate with 500 μl/well of incomplete RPMI with 250 mg/mL of Liberase (Roche, Cat #05401054001) and 10 mg/mL of DNase I (Sigma-Aldrich, Cat #4536282001) for 90 min (ear) or 120 min (footpad) at 37 °C, 5% CO2. The enzymatic reaction was stopped with 1 mL of RPMI supplemented with 10% FBS. The digested tissues were dissociated using a cell strainer (40 mm) in PBS containing 0.05% BSA and 20 mM EDTA (Invitrogen, Cat #130-104-075). pdLNs and spleen were homogenized using a cell strainer (40 μm Falcon). Red blood cells were lysed when required. Cells were washed for 5 min at 1200 rpm to obtain single-cell suspensions.

ELISA

Splenocytes were incubated for 96 hours with 5 nM PEPCK peptide (Selleckchem) and supernatants were frozen at −20 °C until assayed by ELISA. IFN-γ production was assessed using a Immulon 2HB flat-bottom 96-well plate (ThermoFisher, 3855) coated with 0.5 mg/mL anti-mouse IFN-γ clone AN-18 (eBioscience, 14-7313-85) in 1X dPBS at 4 °C overnight. Standards were prepared by diluting recombinant IFN-γ (Biolegend, 575308) in assay media at a max concentration of 1000 pg/mL with 2-fold serial dilutions. Samples were added at the appropriate dilution in assay media and incubated for 120 min at 37 °C. 100 μL of biotinylated anti-mouse IFN-γ clone R46A2 (eBioscience, 13-7312-85) in 1x dPBS + 5% NCS was added at a concentration of 1 μg/mL and incubated for 60 min at 37 °C. Peroxidase-conjugated Streptadivin (Jackson-Immuno Research, 016-030-084) diluted 1000-fold in 1x dPBS + 5% NCS was added at 100 μL/well. The plate was developed using 100 μL of ABTS peroxidase substrate (SeraCare, 5120-0041) per well. The reaction was read at 405 nm. Between each step, the plate was washed at least 5 times with 1X dPBS containing 0.05% Tween 20 (Sigma®). Only samples with PEPCK antigen stimulation were diluted four times, and this dilution factor was used when calculating final IFN-γ levels.

Flow cytometry staining

For intracellular staining, single-cell suspensions were stimulated in vitro with PMA (50 ng/mL), ionomycin (500 ng/mL; Sigma-Aldrich, St Louis, MO, USA), and brefeldin A (BioLegend, San Diego, CA, USA) for three hours. Cells were washed in FACS buffer, and the pellet was further washed and stained using the Foxp3/Transcription factor Fixation/Permeabilization kit (eBioscience™). Surface staining in FACS buffer. The antibodies used were: CD45.2 (104, eBiosciences), CD69 (H1.2F3, eBiosciences), CD150 (TC15-12F12.2, eBiosciences), CD4 (RM4-5, eBiosciences), CD62L (MEL-14, eBiosciences), CD186/CXCR6 (SA051D1, eBiosciences), PD-1 (29F.1A12, eBiosciences), CD44 (IM7, eBiosciences), CD54 (YN1/1.7.4, eBiosciences), CD43 (1B11, eBiosciences), CXCR5 (L138D7, eBiosciences), IFN-γ (XMG1.2, eBiosciences), T-bet (4B10, eBiosciences), Foxp3 (FJK-16s, eBiosciences), Purified Mouse P-Selectin - IgG Fusion Protein (R&D) and Recombinant Mouse E-Selectin/CD62E Fc Chimera Protein (R&D). Cell acquisition was performed on a BD FACSymphony™ A3 flow cytometer (BD Biosciences, San Jose, CA, USA), and the data were processed using FlowJo software (Tree Star, Ashland, OR, USA). For cell counting from skin tissue, AccuCount fluorescent particles were added before flow cytometry acquisition (Spherotech, Lake Forest, IL, USA).

Assessment of parasite burden

Ear cell suspensions were centrifuged at 3000 rpm for 10 min. The cell pellet was resuspended in 400 μL of completed Schneider’s insect medium and used for parasite titration. LNs were collected, and single-cell suspensions were used for parasite quantitation. The homogenates were serially diluted (1:5) in 96-well plates and incubated at 26 °C. The number of viable parasites was calculated from the highest dilution at which parasites were observed after 7 days of incubation.

UMAP analysis

The FlowJo UMAP plug-in version 4.0.386 was used to generate UMAP projections of flow cytometry data. Prior to UMAP analysis, keywords were used to label features of interest for each sample (for example, target cell population, tissue, immunization) to preserve them during concatenation, and populations of interest were concatenated using FlowJo’s concatenate function. UMAP analysis was then run using all parameters except those used for negative selection.

Statistical analysis

Statistics were performed using GraphPad Prism Version 10.2.2 statistical analysis software. For normally distributed data, comparing two groups along a single variable, a standard Student’s t test was used. A Mann–Whitney Rank Sum Test was used if the data was abnormally distributed. When comparing two groups along more than one variable, an Ordinary One-Way ANOVA with a Dunnett’ s multiple comparisons test was used. P-values less than 0.05 were considered significant.