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

Abstract

Dermal resident memory CD4⁺ T cells (dTrm) provide protection 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.

Fig. 1. PEPCK mRNA-LNP vaccine induces a systemic T cell response but not protection against Leishmania major.

A Experimental schematic showing adoptive transfer of T cells from PEPCK-specific TCR transgenic mice (Ptg) cells (CD45.2+) into congenically distinct CD45.1⁺ hosts on day 0 (d0), followed by subcutaneous immunization with 2 μg PEPCK mRNA-LNP on day 1 (d1). At 1-wk post immunization, flow cytometry analysis of donor Ptg cells was performed in the B popliteal draining lymph node (pdLN) and E spleen. C Representative flow cytometry plots showing donor Ptg cells from the pdLN expressing CD44 vs. CD62L and PD-1^hi vs. CXCR5 and D summary plots showing absolute numbers of each T cell subset. F Representative flow cytometry plots of donor Ptg cells from the spleen expressing CD44 vs. CD62L, PD-1^hi vs. CXCR5, and T-bet vs. IFN-γ. G Summary plots showing absolute numbers of each T cell subset in the spleen. H Experimental schematic showing subcutaneous immunization with 2 μg PEPCK mRNA-LNP. I–N Same analysis as (B–G) but with endogenous cells stained with PEPCK I-Ab tetramer. O IFN-γ production by splenocytes from naïve or immunized mice stimulated with or without PEPCK peptide for 96 h. P Schematic showing the L. major infection model. WT mice were either naïve (no immunization), immune (recovered from infection), or immunized subcutaneously with 2 μg PEPCK mRNA-LNP 6 weeks prior to infection. Ear thickness was measured to record Q lesion size weekly for 4-weeks post-infection and R delayed-type hypersensitivity responses (DTH) in the first 48- and 72-h post-infection. Both schematic images were created with Biorender.com. Data from at least two independent experiments, n = 3–5 per experiment. Error bars indicate SD; ****P < 0.0001.

Fig. 2. PEPCK mRNA-LNP vaccine promotes the expression of skin-homing molecules and migration of CD4⁺ T cells to inflamed and non-inflamed skin at 1 week post-immunization.

A Flow cytometry analysis of P and E selectin ligands assessed by P and E selectin Fc reagents (referred to as PESL-Fc) or CD43/1B11 expression on Ptg cells in the popliteal draining lymph node (pdLN) and B spleen at 1-week post subcutaneous immunization with 2 μg PEPCK mRNA-LNP. C Absolute numbers of PESL-Fc⁺ and CD43/1B11⁺ in each tissue. D Representative plot of PD-1^hi vs. CXCR5 expression on donor Ptg cells in the pdLN, histograms and frequencies of CD43/1B11⁺ cells in CXCR5⁺ and CXCR5⁻ subsets. E Same analysis as (D) performed on spleen cells. Representative flow cytometry plots of donor Ptg cells in F inflamed (IS) and G non-inflamed skin (NIS) at 1-week post-immunization. H Frequencies and absolute numbers of donor Ptg cells in inflamed and non-inflamed skin. I Representative PD-1^hi vs. CXCR5 expression plots on donor Ptg cells in IS and NIS. Data from two independent experiments, n = 3 per experiment. Error bars indicate SD; ***P = 0.0004; ****P < 0.0001.

Fig. 3. PEPCK mRNA-LNP vaccine fails to globally seed CD4⁺ dermal Trm cells.

A Experimental schematic showing subcutaneous immunization of WT mice with either 2 or 5 μg PEPCK mRNA-LNP. B Representative flow cytometry plots and C total cell numbers of PEPCK I-Ab Tetramer CD4⁺ T cells in inflamed (IS) and non-inflamed skin (NIS) after 3 weeks of either immunization dose. D Schematic showing adoptive transfer of T cells from PEPCK specific TCR transgenic mice (Ptg) cells (CD45.2+) into congenically distinct CD45.1⁺ hosts on day 0 (d0), subcutaneous immunization with either 2 μg PEPCK mRNA-LNP or peptide vaccine on day 1 (d1) and boost on day 8 (d8). E Representative flow cytometry plots comparing donor Ptg cells in IS and NIS after PEPCK mRNA or peptide vaccine and F total donor cell numbers after 4-weeks of either vaccine. Both schematic images were created with Biorender.com. Data from two independent experiments, n = 3–5 per experiment. Error bars indicate SD; ***P ≤ 0.0006.

Fig. 4. Codelivery of IL-12 mRNA-LNP with PEPCK mRNA-LNP vaccine promotes and maintains CD4⁺ dermal Trm cells in non-inflamed skin.

A Systemic (spleen) frequency and total cell number of donor Ptg cells expansion at 1-week post-subcutaneous immunization with either 2 μg PEPCK mRNA-LNP (blue circles), 1 μg PEPCK mRNA-LNP + 1 μg empty-LNP (blue triangles) or 1 μg PEPCK mRNA-LNP + 1 μg IL-12 mRNA-LNP (orange triangles). B Frequency of donor Ptg cells in blood at days 7, 14, 21 and 28 post-immunization with 1 μg PEPCK mRNA-LNP + 1 μg empty-LNP (blue triangles) or 1 μg PEPCK mRNA-LNP + 1 μg IL-12 mRNA-LNP (orange triangles). UMAP projections of high parameter flow cytometry data of Ptg cells in the (C) spleen at 1-week postimmunization, the source of donor Ptg cells overlayed on the projection. D The expression level of each indicated marker is overlayed on the UMAP projection, E histogram, and F frequency of indicated marker by donor Ptg cells from their respective host in the spleen. G–J Same analysis as the spleen but for popliteal draining lymph node (pdLN). K Representative flow cytometry plots comparing donor Ptg cells and total cell numbers in inflamed (IS) and noninflamed skin (NIS) after PEPCK mRNA-LNP or PEPCK + IL-12 mRNA-LNP at 1-week postimmunization. L Same analysis as (K) at 4-weeks post-immunization. 1-week data from four independent experiments, and 4-weeks data from three independent experiments, n = 3–5 per experiment. Error bars indicate SD; **P = 0.007; ***P = 0.0002; ****P < 0.0001.

Fig. 5. Codelivery of IL-12 mRNA-LNP with PEPCK mRNA-LNP vaccine induces protective immunity to L. major.

A Schematic showing the L. major infection model: adoptive transfer of T cells from PEPCK specific TCR transgenic mice (CD45.2+) into congenically distinct CD45.1⁺ hosts on day 0 (d0), which were either naïve (no immunization) or immunized subcutaneously with 1 μg PEPCK mRNA-LNP + 1 μg empty-LNP (blue triangles) or 1 μg PEPCK mRNA-LNP + 1 μg IL-12 mRNA-LNP (orange triangles) 4-weeks before infection. Ear thickness was measured to assess (B) delayed-type hypersensitivity responses in the first 48- and 72-h post-infection, (C) lesion size and (D) pathology score were recorded weekly for 4-weeks post-infection. Parasite burden was quantified in the (E) infected ear and draining lymph node at 4-weeks post-infection. F Schematic showing the L. major infection model of WT mice that were either naïve (no immunization) or immunized subcutaneously with 1 μg PEPCK mRNA-LNP + 1 μg empty LNP (blue triangles) or 1 μg PEPCK mRNA-LNP + 1 μg IL-12 mRNA-LNP (orange triangles) 4 weeks before infection. Ear thickness was measured to assess (G) delayed-type hypersensitivity responses in the first 48- and 72-h post-infection and (H) lesion size was recorded weekly for 4-weeks post-infection. Parasite burden was quantified in the (I) infected ear and draining lymph node at 4-weeks post-infection. Both schematic images were created with Biorender.com. Data from three independent experiments, n = 5 mice per group. Error bars indicate SD; *P = 0.05; **P 0.002; ****P < 0.0001.