mRNA vaccines engage unconventional pathways in CD8 T cell priming

Abstract

Vaccines composed of mRNA and lipid nanoparticles (LNP) activate B and T cells by inducing in vivo production of specific protein antigens. While B cells can be activated directly by antigens, T cell activation requires antigen processing and presentation by MHC molecules on specialized antigen presenting cells (APCs). In response to viral infections, tumors, and protein- and cDNA-based vaccines, antigen presentation to CD8 T cells is particularly dependent on type 1 conventional dendritic cells (cDC1s) which are specialized for efficient cross-presentation of exogenous antigens^1-4. However, whether similar mechanisms are employed by mRNA-LNP vaccination is currently unknown. We report here that mRNA-LNP vaccines do not require cDC1s or the WDFY4-dependent cross-presentation pathway for CD8 T cell priming but instead engage both cDC1s and cDC2s redundantly. While CD8 T cells primed exclusively by either cDC1 or cDC2 showed phenotypic differences, both could mediate anti-tumor responses and memory formation. Importantly, acquisition by cDCs of peptide-MHC-I complexes from non-hematopoietic cells, called cross-dressing, provides a substantial component of CD8 T cell priming, in a manner dependent on type 1 interferon. mRNA-LNP induction of cross-dressing might explain their capacity to activate CD8 T cells against antigens not encoded by the vaccine.

Introduction

Messenger RNA (mRNA) vaccines delivered by lipid nanoparticle (LNP) were adopted for widespread use in response to the global SARS-CoV-2 pandemic^5-7. The ability of mRNA to be delivered directly into cells and translated into proteins was first demonstrated in vitro using liposomes⁸. Subsequent studies showed the ability to deliver mRNA into cells in vivo and proposed the potential for use in vaccines^9,10. Indeed, mRNA vaccines encoding the influenza hemagglutinin (HA) induced protective antibody responses in mice¹¹. This and subsequent studies found that these vaccines also induced CD4 and CD8 T cell responses in mice¹² and non-human primates¹³. Likewise, mRNA-LNP vaccines to SARS-CoV-2 also induced CD8 T cell responses in humans that appeared to contribute to protection¹⁴.

Due to their apparent success against SARS-CoV-2, mRNA vaccines are now being tested for use in inducing anti-tumor CD8 T cell responses in humans¹⁵. Initial studies in mice predating the SARS-CoV-2 pandemic showed that mRNA vaccines could induce CD8 T cell responses against model antigens expressed by tumors¹⁶. Ongoing clinical trials are testing their value for inducing responses to neoantigens in pancreatic ductal adenocarcinoma (PDAC)¹⁵. Importantly, CD8 T cell priming against foreign antigens can proceed by several different antigen processing pathways and can involve distinct types of antigen presenting cells (APCs), each of which are influenced by the form of antigen delivered^17,18. However, these aspects of CD8 T cell priming have not been evaluated in the context of mRNA vaccination.

Conventional dendritic cells (cDCs) are the primary APCs responsible for priming naive T cells and comprise the type 1 cDC (cDC1) and cDC2 subsets^19,20. The cDC1 subset is the principal APC responsible for capture and presentation of cell-associated antigens in response to tumors, viral infections, and allografts^1,2,21. Likewise, studies on cDNA- and protein-based vaccines have demonstrated that the cDC1 is required for vaccine-induced CD8 T cell priming^3,4. However, these issues have not been addressed for mRNA vaccines. Importantly, the identity of the APC that primes T cells can significantly influence the effectiveness of the response to vaccination. For example, rejection of a murine fibrosarcoma T3 can be induced by vaccination with peptides containing epitopes for MHC I- and MHC II-restricted neoantigens²². At effective doses, cDC1 is the APC responsible for presentation to both CD4 and CD8 T cells. However, higher doses of the MHC II-restricted peptide led to presentation by cDC2 and induced CD4 T cells to acquire a suppressive type 1 regulatory²³ (Tr1) phenotype that caused a failure in T3 rejection²². In short, there is strong motivation to examine the basis by which mRNA vaccines induce CD8 T cell responses.

Recent studies using PET-CT and bioluminescence imaging have investigated the biodistribution of mRNA-LNP vaccines, demonstrating mRNA and protein expressions predominantly occurs at the injection site and draining lymph nodes (dLNs)^24-27. Some studies reported that there was much less antigen expression found at distal sites such as spleen and liver^25,26 compared to the injection site, suggesting that immune priming might occur only at local sites of immunization. However, these studies did not evaluate the immunogenicity of the low levels of antigen produced at distal sites, leaving this issue unresolved. Therefore, there is additional motivation to carry out functional biodistribution analysis that examines the immunological impact of antigen trafficking following mRNA-LNP vaccination.

Here, we used recently developed mouse models^28,29 of cDC subset deficiency to test the requirement for cDC1, cDC2 and cross-presentation in CD8 T cell responses to mRNA-LNP vaccines. We found that mRNA-LNP vaccines engage several diverse and complementary pathways of antigen presentation for the induction of CD8 T cell responses. In contrast to cDNA- and protein-based vaccines, which rely on the cDC1 subset^3,4, mRNA vaccines induce systemic CD8 T cell responses independently of cDC1. Rather, the cDC2 subset plays a more prominent role in priming CD8 T cell responses both ex vivo and in vivo. Surprisingly, mRNA vaccines engaged cDCs in a way that does not require self MHC-I expression. Instead, cDCs acquired non-self MHC-I complexes from non-hematopoietic cells, a process called cross-dressing^30,31. Notably, multimodal single-cell analyses of antigen-specific CD8 T cells primed by cDC1 vs. cDC2 revealed notable differences in clonal proliferation but generally similar effector differentiation consistent with the observed tumor rejection by mRNA vaccination induced in both cDC1- and cDC2-deficient mice.

Results

Systemic CD8 T priming in mRNA vaccine

Previous studies suggested that mRNA-LNP vaccination induces antigen primarily in the injected muscle and dLNs, but reported minimal or undetectable antigen expression at distant organs in mice and non-human primates^24-27. However, since CD4 and CD8 T cells can respond to as little as a few hundred peptide-MHC complexes^32-34, we examined the functional biodistribution of antigens induced by mRNA-LNP vaccination. For this, we measured OT-I proliferation across various lymphoid tissues distal to the intramuscular injection site of 10 μg OVA mRNA-LNP, a dose comparable to that used in previous biodistribution studies (Extended Data Fig. 1 a-d and 2 a, b). Naïve OT-I cells were distributed systemically within 24 hours of adoptive transfer, populating peripheral blood and all lymphoid tissues examined. Two days after immunization, OT-I cells were depleted from blood circulation (Extended Data Fig. 1 b and 2 b), consistent with the reported rapid sequestration of antigen-specific lymphocytes into sites of antigen encounter³⁵. By day 3, proliferating OT-I cells had reappeared in the blood. Notably, OT-I cells underwent comparable divisions in spleen, blood and LNs (Extended Data Fig. 1 b-d). Further, a 100-fold reduction in OVA mRNA-LNP (0.1 μg) induced OT-I proliferation at distant sites at levels similar to that in dLNs (Extended Data Fig. 2 c). On day 1, even before cell division occurred, CD69 was upregulated in OT-I cells in all lymphoid tissues examined, confirming that CD8 T cell priming occurred systemically (Extended Data Fig. 2 d, e). Collectively, these observations suggest that intramuscular vaccination induced CD8 T cell priming in multiple lymphoid tissues in a systemic manner.

To test whether OT-I proliferation observed in distal tissues involved migration of OT-I cells initially activated at the injection site, we treated mice with FTY720 6 hours prior to vaccination to prevent S1PR1-dependent lymphocyte egress³⁶ and then reevaluated OT-I proliferation following mRNA-LNP vaccination (Extended Data Fig. 1 e and 3 a-c). As a positive control, FTY720 treatment prevented the recirculation of dividing OT-I cells into the blood (Extended Data Fig. 1 e and 3 a). Despite this blockade, OT-I proliferation in spleen and LNs 2 and 3 days after intramuscular vaccination was comparable to control mice, respectively (Extended Data Fig. 1 e, and 3 a-c), indicating that T cell activation occurred in response to local antigen presentation.

To test whether cells at the injection site, such as muscle cells or macrophages, could function as APCs to prime CD8 T cells, we first tested the requirement for naïve T cell entry into lymphoid tissues (Extended Data Fig. 1 f and 3 d-f). CD62L blockade inhibits naïve T cell entry into LNs but not spleen³⁷. However, combined CD62L blockade and splenectomy restrict naïve T cell activation to the peripheral tissues³⁸. We therefore evaluated OT-I proliferation induced by OVA mRNA-LNP in splenectomized mice treated with anti-CD62L antibody. Notably, OT-I expansion was significantly reduced in splenectomized mice treated with anti-CD62L on day 3 after immunization (Extended Data Fig. 1 f), indicating that OT-I cells are required to enter lymphoid organs for their priming. In addition, we confirmed that anti-CD62L treatment of splenectomized mice abrogated OT-I accumulation at the injection site (Extended Data Fig. 3 d, f). These results exclude direct CD8 T cell priming by cells at the injection site and reinforce that priming occurs within secondary lymphoid tissues following mRNA vaccination.

Both cDC1 and cDC2 prime CD8 T cells

To identify the APCs responsible for priming T cells after mRNA-LNP vaccination, we purified cDC1, cDC2, and B cell from draining and contralateral LNs on day 2 after vaccination (Fig. 1 a-c, Extended Data Fig. 4a). As expected, cells from unimmunized mice failed to induce OT-I proliferation. However, both cDC1 and cDC2, but not B cells, isolated from dLNs were able to induce OT-I cell proliferation. By contrast, from contralateral LNs, only cDC2 could induce OT-I proliferation (Fig. 1 a, c). The weaker OT-I proliferation induced by cDC2 from contralateral LNs is consistent with lower antigen availability at distant sites following mRNA-LNP vaccination, as described^24-27. These results agree with results shown in Extended Data Fig. 1 that distant sites can be functionally immunogenic.

Figure 1. Both cDC subsets, cDC1 and cDC2, prime CD8 T cells after mRNA vaccination.

a-c, ex vivo OT-I proliferation assay. Mice were intramuscularly (i.m.) immunized with 10 μg OVA mRNA-LNP. Two days later, cDC1, cDC2, and B cells were sorted from draining and contralateral lymph nodes (LNs) and co-cultured with CTV (cell trace violet)-labeled OT-I cells for 3 days. a, Representative flow plots. b, Experimental schematic. c, Quantification of proliferating OT-I. (n=3 unimmunized, n=6 dLN, n=5 cLN.) Two-tailed multiple t-tests. P***=0.00048, P**=0.0095, ns=0.2094. d, e, WT, Δ32, and Δ1+2+3 mice were injected on days 0 and 7 with control or OVA cDNA. Spleens were analyzed on day 11 for SIINFEKL–Kb tetramer+ CD8 T cells and CD44/CD62L expression. (control cDNA: n=1-2; OVA: n=8 WT, n=6 Δ32, n=7 Δ1+2+3.) One-way ANOVA with Tukey’s multiple comparisons. f, g, Mice were immunized with dead or OVA mRNA-LNP on days 0 and 7, and analyzed on day 11. f, Representative flow plots. g, Quantification of tetramer+ CD44+ CD62L+ CD8 T cells. (Control mRNA: n=5 WT; OVA: n=9 WT, n=11 Δ32, and n=11 Δ1+2+3.) One-way ANOVA with Tukey’s multiple comparisons. h-j, in vivo OT-I proliferation assay. OT-I cells were transferred to WT, Δ32, or Δ1+2+3 mice, followed by OVA mRNA-LNP. Spleens were analyzed 2 days later. (control: n=2; OVA: n=6 per genotype.) h, Representative flow plots. i, Quantification of proliferating OT-I. One-way ANOVA with Tukey’s multiple comparison. j, Division distribution among proliferating cells. Two-way ANOVA. k-m, Tetramer response on day 7 after OVA mRNA-LNP immunization in CD11c-DTR (k, l) and CD11c-DTR bone marrow (BM) chimera (m), with or without DT treatment. One-way ANOVA with Tukey’s multiple comparison. l, P***(left)=0.0005, P***(right)=0.0004, ns=0.9584. m, P**=0.0071. n, Tetramer response in Δ32 and Δ32xΔ1+2+3 (DKO). (Unimmunized: n=3; immunized: n=7 Δ32, n=8 DKO.) One-way ANOVA. ns=0.7789. Data are represented as mean values ±s.d. of pooled biologically independent samples from two to four independent experiments.

The robust in vitro activation of OT-I cells by cDC2 (Fig. 1 a-c) was surprising, given that cDC1 are the primary APC responsible for priming CD8 T cells^2-4. To investigate this phenomenon in vivo, we examined endogenous CD8 T cell responses in mice selectively lacking cDC1 or cDC2. For cDC1 deficiency, we used the Irf8 +32^−/− (Δ32) model²⁸. For a cDC2-deficient model, we employed Δ1+2+3 mice²⁹, which harbor triple mutations in the C/EBP binding sites of the −165 kb Zeb2 enhancer and consequently lack cDC2.

First, we immunized WT, Δ32, and Δ1+2+3 mice with an OVA-encoding cDNA vaccine and measured CD8 T cell responses using K^b-SIINFEKL tetramer staining (Fig. 1 d, Extended Data Fig. 4 b) and IFN-γ ELISpot following SIINFEKL restimulation (Extended Data Fig. 4 c). WT and Δ1+2+3 mice generated robust CD8 T cell responses in both assays, while in contrast, Δ32 mice completely lacked tetramer⁺ and IFN-γ⁺ CD8 T cells. This result confirms the strict requirement and sufficiency for cDC1 in priming CD8 T cells in response to cDNA vaccine. In addition, we confirmed a similar cDC1 requirement for CD8 responses to AddaVax-adjuvanted OVA protein vaccine⁴ (Extended Data Fig. 4 d, e). In summary, cDNA- and protein-based vaccines show a strong dependence on cDC1 for effective CD8 T cell priming.

We next asked if mRNA-LNP vaccination requires cDC1 to induce CD8 T cell responses (Fig. 1 f, g). We immunized WT, Δ32, and Δ1+2+3 mice with OVA-encoding mRNA-LNP vaccine and measured the induced CD8 T cell response using K^b-SIINFEKL tetramer staining, using “dead” non-coding mRNA-LNP as a negative control (Fig. 1 f, g). In contrast to cDNA- and protein-based vaccines, the mRNA-LNP vaccine-induced CD8 T cell responses in cDC1-deficient Δ32 mice were comparable to WT mice. Rather, Δ1+2+3 mice showed moderately reduced tetramer responses, approximately 60% of WT, in agreement with superior ex vivo presentation by cDC2 (Fig. 1 a-c). Lastly, to test whether this result extends beyond OVA antigen, we used an mRNA vaccine encoding tumor neoantigen, a mutant peptide from Lama4 (mLama4) and observed comparable tetramer expansion that WT, Δ32, and Δ1+2+3 mice (Extended Data Fig. 4 h). Thus, mRNA-LNP vaccination induces strong CD8 T cell responses using either cDC1 or cDC2 as an APC.

Tetramer responses measured on day 11 reflect cumulative outcomes of priming, expansion, survival, and trafficking. Therefore, we directly examined early CD8 T cell priming and proliferation in response to mRNA-LNP vaccination. For this, we transferred CTV-labeled OT-I cells into WT, Δ32, and Δ1+2+3 mice and immunized with OVA mRNA-LNP vaccine. On day 2, OT-I cells in all genotypes proliferated with comparable number of divisions (Fig. 1 h-j), suggesting that mRNA-LNP vaccination can employ cDC1 and cDC2 redundantly to prime CD8 T cells.

cDCs are the primary APCs that prime naïve T cells. To validate that cDCs are required for CD8 T cell priming in mRNA vaccination, we assessed SIINFEKL-K^b tetramer⁺ CD8 T cell expansion in CD11c-DTR mice following OVA mRNA-LNP vaccination. CD11c-DTR mice were treated with diphtheria toxin (DT) every 2-3 days and vaccinated with OVA mRNA-LNP (Fig. 1 k, l, Extended Data Fig. 5 b-f). We first confirmed a specific and efficient depletion of cDCs after DT treatment (Extended Data Fig. 5 b-e). Importantly, tetramer responses were nearly abrogated in DT-treated CD11c-DTR mice, demonstrating that cDCs are required for CD8 T cell priming following mRNA vaccination (Fig. 1 k, l). In addition, to minimize potential off-target toxicity of DT, we also evaluated the CD11c-DTR → SJL bone marrow (BM) chimera mice. These DT-treated BM chimeras similarly showed loss of SIINFEKL-K^b tetramer expansion (Fig. 1 m, Extended Data Fig. 5 d), with selective depletion of cDCs but not macrophages or inflammatory monocytes (Extended Data Fig. 5 g-i).

Lastly, we found that Δ32 x Δ1+2+3 mice, which lack cDC1 and monocyte development²⁹, have a normal CD8 T cell tetramer expansion in response to mRNA-LNP vaccination (Fig. 1 n). This indicates that monocytes or monocyte-derived cells are not required for cDC1-independent CD8 T cell priming. Together, these data identify cDC1 and cDC2 as the principal APCs responsible for CD8 T cell priming after mRNA vaccination.

Dispensable MHC-I on APC to prime CD8 T

Peptides from antigens encoded by mRNA-LNP could conceivably be loaded onto MHC-I molecules either by direct transfection of APCs³⁹ or by cross-presentation. We have previously shown that the BEACH domain protein WDFY4 is required for cross-presentation of cell-associated antigens⁴⁰ and immune complex antigens¹⁸. However, we find that CD8 T cell responses induced by mRNA-LNP were equivalent in WT and Wdfy4^−/− mice by both tetramer staining and IFN-γ ELISpot (Extended Data Fig. 5 j-l), indicating that cross-presentation is not a substantial pathway in CD8 T cell responses to mRNA-LNP vaccination.

While direct presentation and cross-presentation require APCs to express MHC-I, cross-dressing⁴¹ is an unconventional antigen presentation pathway, in which peptide-MHC-I complexes are transferred from donor cells onto cDCs prior to T cell activation. Importantly, cross-dressing bypasses the requirement for MHC-I expression by cDCs. To test whether cross-dressing is engaged by mRNA-LNP vaccination, we conditionally deleted beta-2-microglobulin (β2m) expression in CD11c⁺ APCs by crossing Itgax-Cre⁺ mice with β2m^fl/fl mice. Itgax-Cre⁺ β2m^fl/fl mice lack surface expression of H2-K^b and H2-D^b on cDCs, but retain expression on other cells including B cells, monocytes and macrophages¹⁸. As expected, these mice failed to prime OT-I cells in response to Abelson-mOVA tumor, which requires cross-presentation¹⁸, similar to global MHC-I TKO mice (Kb^−/− Db^−/− β2m^−/−) (Fig. 2 a, b). However, Itgax-Cre⁺ β2m^fl/fl mice showed robust OT-I T cell priming in response to OVA mRNA-LNP, similar to control β2m^fl/fl mice (Fig. 2 c-e). Furthermore, similar findings were obtained by examining the endogenous T cell repertoire in these mice (Fig. 2 f, g). Itgax-Cre⁺ β2m^fl/fl mice had robust tetramer⁺ CD8 T cell responses with normal CD44 induction, though the frequency of IFN-γ⁺ cells was modestly reduced (~10%) (Fig. 2 f, g, Extended Data Fig. 4 f). Therefore, cDC expression of MHC-I may not be required for CD8 T cell priming after mRNA vaccination, suggesting possible involvement of cross-dressing.

Figure 2. MHC-I on APCs is not required for CD8 T cell priming after mRNA vaccination.

a-e, in vivo OT-I proliferation assay. CTV-labeled CD45.1+ naïve OT-I cells were transferred to b2mfl/fl, Itgax-Cre+; b2mfl/fl, Kb−/−;Db−/−;b2m−/− (MHC-I TKO), or Δ32 mice. One day later, mice were immunized with Abelson-mOVA tumor (a, b) or OVA mRNA-LNP (c-e). Spleens were analyzed 3 days after tumor or 2 days after mRNA-LNP. a, c, Representative flow plots. b, d, Quantification of proliferating OT-I cells. One-way ANOVA with Tukey’s multiple comparisons. b, P***(left)=0.0003, P***(right)=0.0004, ns=0.9710. d, P****<0.0001, ns=0.3009. e, Division distribution among proliferating cells. Two-way ANOVA. (n=5 per genotype) P****<0.0001, ns=not significant. f, g, mice were immunized with OVA mRNA-LNP on days 0 and 7 and analyzed on day 11. f, Representative flow plots. g, Quantification of tetramer+ CD44+ CD62L+ CD8 T cells. (Dead: n=3; OVA: n=8 b2m^fl/fl, n=7 Itgax-Cre+; b2m^fl/fl.) One-way ANOVA with Tukey’s multiple comparisons test. h, i, CD45.1⁺ SJL mice were depleted of NK cells, lethally irradiated, and reconstituted with CD45.2⁺ WT or MHC-I TKO BM. After 8 weeks, mice were immunized with OVA mRNA-LNP and analyzed on day 11. h, Schematic (left) and representative flow plots (right). i, Quantification of tetramer+ cells. (unimmunized/dead: n=1-2; OVA: n=6 per genotype). Two-tailed t-tests. P**(left)=0.0026, P*(right)=0.0151. j-l, WT or MHC-I TKO recipients were irradiated and reconstituted with SJL or Δ32 BM. 8 weeks later, mice received CTV-labeled naïve OT-I cells and immunized with OVA mRNA-LNP. j, Schematic. k, Representative flow plots. (n=6, 8, 7, and 6 from left to right.) Two-tailed t-tests. l, Quantification of tetramer+ CD8 T cells. t-test. m, For IFNAR1 blockade, mice were injected i.p. with 2 mg of anti-IFNAR1 on day −1 and 6, one day before each OVA-mRNA immunization. (dead mRNA: n=2; OVA: n=5 per genotype.) Two-tailed t-test. P***=0.0005. Data are represented as mean values ± s.d. and biologically independent samples from two independent experiments.

While Itgax-Cre mediated deletion of β2m is sufficient to abrogate cross-presentation of tumor antigens (Fig. 2 a, b), we previously reported detectable MHC-I expression on some cDCs¹⁸. Furthermore, β2m could potentially be captured from serum⁴² to stabilize MHC-I complexes. To ensure complete absence of MHC-I expression on APCs, we generated BM chimeras using WT or MHC-I TKO donors into irradiated CD45.1⁺ recipient mice depleted of NK cells (Fig. 2 h). We confirmed complete donor-derived chimerism (99.8-100%) for peripheral cDC in spleen and MDP, CDP, pre-cDC1, and pre-cDC2 in BM (Extended Data Fig. 6 a-d). Next, we tested these BM chimeras for CD8 T cells responses following mRNA-LNP vaccination. In B6 → SJL control chimeras, mRNA-LNP vaccination induced strong expansion of tetramer+ CD8 T cells (~13%) (Fig. 2 h, i). Surprisingly, MHC-I TKO → SJL chimeras, in which cDCs completely lack self-MHC-I, still generated significant tetramer⁺ CD8 T cells (~5%), indicating that MHC-I expression on cDCs is not required for CD8 T cell priming in mRNA-LNP vaccination.

We next sought to quantify the contribution of cross-dressing to CD8 T cell activation in vivo, when other presentation pathways remain available. We generated BM chimeras by transferring WT or Δ32 BM into SJL or MHC-I TKO recipient mice and measured OT-I proliferation following OVA mRNA-LNP vaccination (Fig. 2 j, k). In WT → TKO chimeras, cDCs cannot acquire peptide-MHC-I from non-hematopoietic cells, but retain capacity for direct presentation and cross-presentation. Notably, these chimeras showed reduced OT-I proliferation compared to WT → SJL controls, indicating cross-dressing contributes measurably to strong CD8 T cell response. In Δ32 → TKO chimeras, cDC1 are absent and cDC2 are the sole APC supporting CD8 T cell priming. Importantly, in Δ32 → TKO chimeras, we observed much more substantial reduction in OT-I proliferation compared to the Δ32 → SJL controls. This indicates that cross-dressing is a major pathway used by cDC2 for CD8 T cell priming in mRNA-LNP vaccination.

A recent study found that cross-dressing by cDC2 in a tumor relies on type I interferon signaling⁴³, a pathway strongly activated by mRNA vaccines⁴⁴. Thus, we vaccinated Δ32 mice with mRNA-LNP with or without administration of blocking anti-IFNAR1 antibodies (Fig. 2 l, m, Extended Data Fig. 7 a-d). Anti-IFNAR1 treatment significantly decreased tetramer⁺ CD8 T cells in Δ32 mice. Importantly, we observed that anti-IFNAR1 also reduced transfer of H2-K^b to cDCs in Itgax-Cre⁺ β2m^fl/fl mice (Extended Data Fig. 7 e, f). Together, these results suggest that mRNA-LNP vaccination induces cDCs to acquire peptide-MHC-I complexes from non-hematopoietic cells in a type I IFN-dependent manner, which functionally support CD8 T cell priming.

mRNA vaccination engages cross-dressing

The results above suggest that cDCs acquire peptide-MHC-I complexes from non-hematopoietic cells during mRNA-LNP vaccination. Indeed, we detected some surface K^b expression on CD45.2⁺ MHC-I TKO cells in vaccinated MHC-I TKO→SJL chimeras but not in unvaccinated controls (Extended Data Fig. 8 a). To rigorously test MHC-I transfer from non-hematopoietic cells to cDCs, we generated fully allogeneic BM chimeras between B6 (H-2^b) and BALB/c (H-2^d) mice in all combinations (Fig. 3 a). This approach allows for detection of distinct H-2K alleles on donor-derived cDCs in in vivo settings. Complete donor-derived chimerism for all peripheral cDCs and BM progenitors was confirmed (Fig. 3 b, Extended Data Fig. 8 b-d). Notably, in spleens of immunized BALB/c→B6 chimeras, cDC1 and cDC2 expressed H2-K^d at high levels and acquired H2-K^b at low levels (Fig. 3 b-e, Extended Data Fig. 8 e, f) in response to OVA mRNA vaccination. In these chimeras, about 10% of cDC1 and 20% of cDC2 were H2-K^d high and H2-K^b positive (Fig. 3 d). This acquisition was specific to the recipient haplotype, as H2-K^b transfer was not observed in BALB/c→BALB/c chimeras (Fig. 3 d-e).

Figure 3. cDCs acquire MHC-I from non-hematopoietic cells and prime CD8 T cells.

a-e, B6 and BALB/c BM cells were depleted of T cells with anti-CD4 and anti-CD8a antibodies and injected into lethally irradiated B6 or BALB/c mice. 8 weeks later, mice were immunized with 10 μg OVA mRNA-LNP and examined for chimerism, MHC-I expression, and tetramer expansion. a, Schematic diagram of experimental setup. b, Representative flow plots of splenic cDC1 and cDC2 from BALB/c → B6, BALB/c → BALB/c, and B6→B6. c, Histograms of cDC1 and cDC2 of H2-K^b expression. Numbers indicate MFI of H2-K^b with full staining. Data are representative of two independent experiments. Two-way ANOVA with multiple comparisons test. d, Quantification of K^b+ K^d+ cells as a percentage of K^d+ cells derived from BALB/c BM. One-way ANOVA with multiple comparisons. P**** < 0.0001. e, Quantification of H2-K^b surface expression of K^d+ cells from either BALB/c or B6 recipient mice. (n=5 for BALB/c→BALB/c, n=9 for BALB/c→B6 mice.) One-way ANOVA with Tukey’s multiple comparisons test. P**** < 0.0001. Data are represented as mean values ± s.d. from pooled biologically independent samples from two independent experiments. ns = not significant.

mRNA-LNP vaccination of fully allogeneic chimeras revealed that cross-dressing can support CD8 T cell priming (Extended Data Fig. 8 g, h). T cell selection in allogeneic chimeras is complicated, generating a T-cell repertoire distinct from both donor and recipient strains^45,46. Nonetheless, OVA mRNA vaccination of BALB/c → B6 chimeras generates an H2-K^b-restricted CD8 T cell response compared to unvaccinated controls (Extended Data Fig. 8 g, h). This is consistent with CD8 T cell priming in MHC-I TKO → SJL chimeras (Fig. 2 h, i), demonstrating that cross-dressing can functionally support CD8 T cell priming.

cDC2 can promote anti-tumor responses

Normally, mice lacking cDC1 fail to reject tumors^2,28. Since mRNA-LNP vaccination induced CD8 T cell priming in Δ32 mice, we asked whether these CD8 T cells were functional. We used 1956 fibrosarcoma expressing membrane-bound OVA (1956-mOVA)⁴⁷, whose rejection of requires cDC1-mediated CD4 and CD8 T cell priming. WT and Δ32 mice were vaccinated with dead or OVA mRNA-LNP and challenged with 1956-mOVA on contralateral flank (Fig. 4 a). Following dead mRNA-LNP vaccination, WT mice rejected tumors while Δ32 mice failed to do so, consistent with cDC1-dependent rejection of 1956-mOVA tumors. In contrast, OVA mRNA-LNP vaccination completely prevented 1956-mOVA growth in both WT and Δ32 mice (Fig. 4 b, c) suggesting that CD8 T cells primed by cDC2 are functionally competent.

Figure 4. CD8 T cells primed by cDC2 are capable of anti-tumor response.

a-c, WT and D32 mice were intramuscularly immunized with 10 μg of either dead or OVA mRNA-LNP on day 0 and day 7, followed by subcutaneous injection of 1e6 1956-mOVA fibrosarcoma on day 11. Tumor size was measured every 3-4 days from day 16. a, Schematic diagram of experimental setup. b, Tumor growth curves of WT and D32 mice pretreated with dead or OVA mRNA-LNP. Data represents biologically independent samples from two independent experiments (n=8 for each condition.) c, Quantification of tumor growth curves. Data represent mean ± s.d. from pooled biologically independent samples shown in b. Two-way ANOVA with Tukey’s multiple comparisons test. P**** < 0.0001. ns = not significant.

In addition to analyzing the effector phase of CD8 T cells, we asked whether cDC1-versus cDC2-mediated priming generates distinct memory CD8 T cell responses (Extended Data Fig. 9). First, we immunized WT, Δ32, and Δ1+2+3 mice on day 0 and 7 with OVA mRNA-LNP, and profiled memory CD8 T cells 5-6 weeks after second dose. All three genotypes exhibited comparable frequencies of SIINFEKL-Kb tetramer⁺ cells (Extended Data Fig. 9 a), which were lower than levels observed on day 11, as expected with effector-phase contraction. Among tetramer⁺ cells, there were higher percentages of CD127⁺ cells in Δ32 mice, compared to Δ1+2+3 mice (Extended Data Fig. 9 b, c). Next, we performed in vivo cytotoxicity assay by transferring 1:1 mixture of irrelevant peptide-loaded (CTV-low) and SIINFEKL-loaded (CTV-high) CD45.1+ splenocytes 6 weeks after second dose. While killing efficiency approached near 100% in WT, D32 and D1+2+3 mice 16 hours after transfer, Δ1+2+3 mice exhibited slightly lower killing efficiency (Extended Data Fig. 9 d, e). This correlated with fewer tetramer⁺ CD8 T cells in spleen and LNs of Δ1+2+3 mice (Extended Data Fig. 9 f, g). Together, these data suggest that both cDC1 and cDC2 generate functional memory CD8 T cells, but may do so with potential quantitative and phenotypic differences.

Distinct CD8 T profiles by cDC1 and cDC2

To further examine whether the identity of the priming cDC impacts the transcriptional and clonal profiles of CD8 T cells, we performed single-cell RNA and TCR sequencing on K^b-SIINFEKL tetramer⁺ CD8 T cells isolated from WT, Δ32, and Δ1+2+3 mice following immunization with OVA mRNA-LNP (Fig. 5, Extended Data Fig. 10). As controls, we included tetramer⁻ CD8 T cells from WT mice immunized with dead or OVA mRNA-LNP. After quality control, we retained single-cell RNA sequencing (scRNA-seq) profiles of 58,461 cells, with paired TCRαβ sequences recovered from 78.7% of these cells. Dimensionality reduction and unsupervised clustering identified 11 distinct populations, reflecting substantial heterogeneity (Fig. 5 a, Extended Data Fig. 10).

Figure 5. Distinct profiles of CD8⁺ T cells primed by cDC1 or cDC2.

a, UMAP visualization of Tet+ and Tet− splenic CD8⁺ T cells from mice at day 11 after immunization with mRNA-LNP on day 0 and 7. UMAP shows 11 distinct clusters from a total of 58,461 cells, colored by immune cell type. b, Dot plot showing expression of cluster-defining marker genes. Eff_Ter: terminal effector; Eff: effector; ExhLike: exhausted-like; ISAG: interferon-stimulated activated genes; Tcm: central memory; Mem: memory. c, Stacked bar plot showing the relative frequency of each immune cell type per mouse, based on cluster representation. d, Bar plot showing clonal abundance within each cluster for individual mice. The plot illustrates how T cell clones are distributed across phenotypic states, highlighting inter-mouse variability in clonal expansion and phenotypic fate. e, Bar plot illustrating the phenotypic distribution of top clones (clone size >20) for each mouse. The upper panel shows log₁₀-transformed clone sizes, and the lower panel represents relative clonal proportions. Each bar corresponds to an individual clone.

Within tetramer⁺ cells, effector subsets were identified by cytotoxic gene expression (Prf1, Granzymes), which further segregated into terminal effectors (S1pr5, Klrg1) and Il7r-expressing effectors (Fig. 5 b). A population of proliferating cells expressing Mki67 shared effector-like features but was distinguished by phase-specific expression of cell cycle genes—Mcm2 (G1 phase), Hist1h2ae (S phase), and Ccnb2 and Cenpa (G2/S phase). We also identified a stem-like subset expressing Tcf7 and Slamf6. In contrast, exhausted-like cells exhibited expressions of Lilrb4a, Lilrb4b, Tox and Cd38, along with inhibitory receptors including Pdcd1 and Lag3. An interferon signaling-associated gene (ISAG) cluster was defined by elevated expression of Isg15, Ifit3, and Ifit1. Among tetramer⁻ cells, we observed both naïve/central memory populations (Sell, Ccr7, Lef1, Tcf7) and memory-like subsets marked by Gzmm and Ifngas1 (Fig. 5 b, c).

Notably, we found that Δ1+2+3 mice had higher proportion of cycling effector cells compared to WT or Δ32 mice (Fig. 5 c). Further, TCR analysis revealed predominance of clonally expanded terminal effector cells in WT and Δ32 mice, in contrast to Δ1+2+3 mice which showed a shift towards cycling and stem-like populations (Fig. 5 d). In addition, hyper-expanded clones (clone size > 20, Supplementary Information Table 1) in Δ1+2+3 mice exhibited more cycling and stem-like features relative to those in WT and Δ32 mice (Fig. 5 e). Together, these findings highlight the phenotypic heterogeneity and clonal diversity of antigen-specific CD8⁺ T cells following immunization, with Δ1+2+3 mice displaying a distinct profile enriched for cycling and stem-like populations, in contrast to the terminal effector-dominated profiles observed in WT and Δ32 mice.

Discussion

Our study makes several novel observations regarding CD8 T cell priming induced by mRNA-LNP vaccines. First, we identify cDC1 and cDC2 as the APCs that redundantly drive CD8 T cell priming in lymphoid tissues, while showing that macrophage/monocyte lineages and somatic cells at the site of injection do not contribute. Second, we show that mRNA-LNP vaccination induces CD8 T cell priming in lymphoid tissues widely throughout the body, despite being delivered to specific intramuscular injection sites. Importantly, we show that a substantial component of the CD8 T cell priming is driven by ‘cross-dressing’, in which peptide-MHC complexes arising from non-hematopoietic cells are directly transferred onto cDCs in a manner dependent on type I interferons. This last observation is particularly significant since it has implications for understanding the recently reported impact of COVID-19 vaccination on anti-tumor responses⁴⁸.

One significant finding of this study is that cDCs, either cDC1 or cDC2, are responsible for priming CD8 T cell responses following mRNA-LNP vaccination. While previous studies showed that antigen can be expressed in diverse cell types at the injection site, including muscle cells and macrophages^26,49, we show that these cells are not responsible for directly priming naïve CD8 T cells. We excluded CD8 T cell priming at the injection site by showing that lymphocyte entry into secondary lymphoid organs was required. Furthermore, despite the high uptake of mRNA-LNP by monocytes and macrophages, these cells were insufficient for CD8 T cell priming in mice lacking cDCs. Further, deletion of monocytes and monocyte-derived cells did not impair CD8 T cell responses, showing they are dispensable for this process. Thus, induction of CD8 T cell responses by mRNA-LNP vaccines relies on the migration of cDCs to from sites of peripheral antigen expression into lymphoid tissues where they can encounter CD8 T cells.

This flexibility of using both cDC1 or cDC2 subsets in priming CD8 T cell responses stand in contrast to other vaccination strategies, such as protein subunits, cDNA-based vaccines or viral vectors and tumors, which are dependent on the cDC1 lineage for cross-presentation^1-4. However, a recent study reported that i.v. delivery of an mRNA-LNP vaccine required cDC1 for inducing CD8 T cell responses which were restrained by type I interferons⁵⁰. It is unclear whether this difference in cDC dependence results from the different route of vaccine delivery, the different lipid composition of the LNP, or differences in the magnitude or quality of the induced systemic cytokine responses. Indeed, i.v. delivery of mRNA-LNP is reported to drive heightened inflammation and tissue delivery to organs including the heart⁵¹. Future studies should address the context-dependent differences that might influence the cDC requirement and role of type I IFN signaling for CD8 T cell priming to mRNA-LNP vaccination.

Another finding was the systemic action of mRNA-LNP vaccines, which may increase the CD8 T cell repertoire recruited into the response. Previous studies of biodistribution primarily focused on the levels of antigen expression across tissues, finding a predominant antigen expression at the injection site, but did not directly assess the immunogenic potential at distant sites. Here, we demonstrate that CD8 T cell priming occurs systemically at multiple lymphoid tissues. Naïve CD8 T cells require ~12 to 20 hours to transit each lymph node before their egress to circulation⁵², which restricts the temporal window in which they can encounter antigens presented locally. Since mRNA-LNP vaccines induce expression of antigen in a transient manner, the systemic priming may enhance their recruitment of reactive T cell repertoire.

The processing pathways used by cDCs in mRNA-LNP vaccination was also unique compared with traditional vaccine platforms. Specifically, in addition to conventional cross-presentation and direct presentation, cross-dressing also contributed a substantial component to the CD8 T cell responses, allowing independence from any particular processing pathway. CD8 T cell priming was independent of WDFY4, a protein required for cross-presentation of cell-associated antigen⁴⁰. Analysis using various BM chimeras revealed that cDCs lacking expression of their own MHC-I proteins were still capable of robustly priming CD8 T cells, indicating capture of pre-formed peptide-MHC-I complexes from other cells. Such cross-dressing of peptide-MHC-I molecules was dependent on type I interferon signaling, in agreement with the reported role of type I interferons on cross-dressing in a tumor model⁴³.

The identity of the APC significantly impacts the character of T cell responses^22,38. For CD4 T cells, priming by cDC1 and cDC2 exerts important differences on the responding T cells that underly effective defenses against pathogens. cDC1, but not cDC2, produce IL-12 and induce protective T_H1 responses in T. gondii infection⁵³, while cDC2, but not cDC1, induce protective T_H2 responses against H. polygyrus²⁹. However, the impact of priming by different cDCs on the quality of CD8 T cells has not been extensively evaluated. In most physiological settings (e.g., viral infections, tumors, conventional vaccines), cDC1, but not cDC2, prime CD8 T cells, preventing a direct comparison of their impact. Recent studies reported that cross-dressing can recruit cDC2 to participate as a minor component of the CD8 T response against tumors^43,54, but did not directly compare CD8 T cells primed exclusively by each subset. However, we were able to directly compare CD8 T cells primed exclusively by cDC1 or cDC2 using cDC1- and cDC2 deficient mouse models. Single-cell transcriptomic analysis reveals some qualitative differences; priming by cDC1 appears to generate hyperexpanded clones with greater capacity for proliferation compared to T cells primed exclusively by cDC2. Priming by cDC2 seems to induce memory cells with somewhat higher IL-7R expression. CD8 T cells primed by cDC2s were capable of mediating anti-tumor responses and generated comparable frequencies of memory T cells which exhibited similar levels of cytotoxicity. However, their effectiveness in settings of chronic responses remains to be evaluated.

Recently, mRNA-LNP vaccines encoding the COVID-19 spike protein were reported to facilitate anti-tumor CD8 T cell responses induced by immune checkpoint blockade in both human and mice⁴⁸. This response was noted to be dependent on type I IFN, but the mechanism underlying the specific response to tumor antigens was unclear. Here, we showed that cross-dressing of peptide-MHC-I complexes by both cDC1 and cDC2 was facilitated by type I IFN following mRNA-LNP vaccination. Importantly, in cross-dressing, the acquisition of peptide-MHC-I complexes is thought to be non-specific to the antigenic peptide that is loaded⁵⁵. Conceivably, mRNA-LNP vaccination could induce cDCs to acquire peptide-MHC-I complexes from other cells, including non-hematopoietic cells or tumors. Together, cross-dressing may broaden the repertoire of antigens that are presented following mRNA vaccination, which may have implications for vaccine efficacy and potential immunopathology involved in mRNA vaccination.

Methods

Mice

WT C57BL/6J (B6) (stock no. 000664), C57BL/6-Tg(TcraTcrb)1100Mb/J (OT-I) (stock no. 003831), B6.FVB-1700016L21RikTg(Itgax-HBEGF/EGFP)57Lan/J (CD11c-DTR) (stock no. 004509), C57BL/6NFWdfy4em1(IMPC)J/J (Wdfy4^−/−) (stock no. 029334), B6.SJL-Ptprc^a Pepc^b/BoyJ (SJL) (stock no. 002014), and BALB/cJ (BALB/c) (stock no. 000651) were obtained from The Jackson Laboratory. OT-I mice were crossed with CD45.1 SJL mice to produce CD45.1 OT-I. Irf8 +32^−/− mice (Δ32) (C57BL/6-Rr253em6Kmm/J, stock no. 032744; The Jackson Laboratory), carrying a homozygous deletion of the +32 kb Irf8 enhancer, Δ1+2+3 mice (C57BL/6-Rr253em6Kmm/J, stock no. 037704; The Jackson Laboratory), carrying homozygous mutations in three CEBPα binding site within the −165 kb Zeb2 enhancer²⁹, and double knockout of Δ32 and Δ1+2+3 (Δ32 x Δ1+2+3) were generated in house and previously described. Mice with floxed β2-microglobulin (β2m^fl/fl) alleles were crossed to Itgax-cre to achieve conditional deletion of β2m on CD11c⁺ cells. MHC-I triple knockout (Kb^−/−Db^−/−β2m^−/−, TKO) mice were generously provided by T. Hansen (Washington University, St Louis, MO)⁵⁶.

All mice were housed in our specific-pathogen free facility with a 12-hour light/dark cycles, maintained at 70 °F and 50% humidity, in compliance with institutional and AAALAC-accredited Animal Studies Committee guidelines at Washington University in St Louis, following all relevant ethical regulations.

BM chimera

For CD11c-DTR BM chimera, CD45.1⁺ SJL recipient mice were lethally irradiated (1,050 rads X-ray). Within 12–18 hours post-irradiation, recipient mice were intravenously injected with ≥ 5×10⁶ BM cells obtained from CD11c-DTR donor mice.

For WT or MHC-I TKO → SJL BM chimera, SJL recipient mice were depleted of NK cells by intraperitoneal injection of 100 μg anti-NK1.1 antibody (clone PK136; Leinco Technologies, cat. N123). The following day, recipient mice were lethally irradiated (1,050 rads X-ray). Within 12–18 hours post-irradiation, recipient mice were intravenously injected with ≥ 5×10⁶ BM cells obtained from either WT or MHC-I TKO donor mice. For WT or Δ32 → SJL or MHC-I TKO BM chimera, SJL or MHC-I TKO recipient mice were lethally irradiated (1,050 rads X-ray). Within 12–18 hours post-irradiation, recipient mice were intravenously injected with ≥ 5×10⁶ BM cells obtained from either WT or Δ32 donor mice.

For B6 and BALB/c allogeneic BM chimera, recipient B6 and BALB/c mice were lethally irradiated with 1,050 rads and 650 rads x-ray, respectively. Donor BM from B6 or BALB/c mice was harvested and treated with ACK lysis buffer to remove erythrocytes. T cells were depleted from donor BM suspensions by incubating cells with biotinylated anti-CD4 (clone GK1.5; BioLegend) and anti-CD8β (clone YTS156.7.7; BioLegend) antibodies, followed by magnetic depletion using MagniSort Streptavidin Negative Selection Beads (Thermo Fisher). Following T cell depletion, ≥ 5×10⁶ cells of prepared BM were intravenously injected into irradiated recipient mice.

All BM chimera recipients were allowed to reconstitute for at least 7 weeks before use in experiments.

Antibodies and flow cytometry

Flow cytometry and cell sorting were performed using either an Aurora flow cytometer (Cytek) or a FACSAria Fusion (BD). Data acquisition was carried out with BD FACSDiva software, and analyses were conducted using FlowJo software version 10.10.0 (BD Biosciences). Surface staining was performed at 4°C in the presence of Fc block (2.4G2) in magnetic-activated cell-sorting (MACS) buffer (PBS supplemented with 0.5% BSA and 2mM EDTA).

For depletion-based sort purification of OT-I T cells and splenic DCs, the following biotinylated anti-mouse antibodies were used: B220 (RA3-6B2), Ly6G (1A8), CD3Ɛ (145-2C11), CD19 (6D5), TER119 (TER-119), CD8β (YTS156.7.7), and CD4 (GK1.5) (all from BioLegend), and CD105 (MJ7/18) (from Invitrogen). Biotinylated cells were detected with BV650-conjugated Streptavidin (BioLegend, 405231) and PE-Cy7-conjugated Streptavidin (BioLegend, 405206).

For biotin- and fluorochrome-conjugated antibodies, the following anti-mouse antibodies were used. From BioLegend: AF488-conjugated B220 (RA36B2; 103225), AF647-conjugated SiglecH (551; 129608), BV510-conjugated I-A/E (M5/114.15.2; 100752), FITC-conjugated KLRG1 (2F1/KLRG1; 138409), PE- and BV421-conjugated XCR1 (ZET; 148204 and 148216), PE-Cy7-conjugated CD24 (M1/69; 138508), APC-Cy7-conjugated Sirpα (P84; 110716), BV605- and BV510-conjugated CD8α (53-6.7; 100751 and 100752), APC-Cy7-conjugated CD45.1 (A20; 110716), PE-Cy7-conjugated CD45.2 (104; 109814), BV421-conjugated H-2Kb (AF6-88.5; 116525), PE-conjugated H-2Db (KH95; 111508), PE-conjugated Vα2 (B20.1; 127808), APC-conjugated CD44 (IM7; 103028 and), PerCP-Cy5.5-conjugated CD62L (MEL14; 104432), biotin-conjugated CD69 (FN50; 310924), FITC-conjugated CD3Ɛ (145-2C11; 100306), FITC-conjugated CD11b (M1/70; 101206), BV711-conjugated CD4 (GK1.5; 100447), AF700-conjugated F4/80 (BM8, 123130), BV421-conjugated Ly6C (HK1.4; 128032), BV711-conjugated CD115/CSF-1R (AFS98; 135515), and APC-conjugated CD226 (10E5; 128810) were used. From BD Biosciences: BUV395-conjugated CD45R/B220 (RA3-6B2), BUV395-conjugated cKit (2B8), BV421-conjugated CD127 (Sb/199), and PE-CF594-conjugated Flt3 (A2F10.1) were used. From Invitrogen: APC-eF780-conjugated CD44 (IM7), APC-eF780-conjugated CD11c (N418), and PerCP-ef710-conjugated Sirpα (P84) were used.

For in vivo IFNAR1 blockade, 2 mg of anti-mouse IFNAR-1 antibody (clone MAR1-5A3; Leinco Technologies, cat. I-401) was administered by intraperitoneal injection every 7 days, beginning one day prior to immunization (day −1 and day 6.)

APC preparation

Lymph nodes were harvested and enzymatically digested in complete IMDM (I10F; Iscove’s modified Dulbecco’s medium with 2ME, NEAA, glutamine, penicillin/streptomycin, and 10% FBS) supplemented with 30 U/ml of DNase I (Sigma-Aldrich) and 250 ug/ml of collagenase B (Roche) for 30-45 min at 37°C. Following digestion, single-cell suspensions were filtered through 70 um strainers, APCs were sorted as B220⁻MHC-II⁺CD11c⁺XCR1⁺CD172α⁻ (cDC1), B220⁻MHC-II⁺CD11c⁺XCR1⁻CD172α⁺ (cDC2), and B220⁺MHC-II⁺ (B cells).

Spleen, lymph nodes, and bone marrow preparation

For cDC staining, spleen and lymph nodes were harvested and enzymatically digested in I10F supplemented with 30 U/ml of DNase I (Sigma-Aldrich) and 250 ug/ml of collagenase B (Roche) for 30-45 min at 37°C. Following digestion, single-cell suspensions were filtered through a 70 μm strainer and stained for flow cytometry analysis.

For CD8 T cell staining, spleen, lymph nodes, and peripheral blood were harvested, mechanically dissociated, and passed through a 70 μm strainer for single-cell suspensions. After ACK lysis, cells were stained for flow cytometry.

BM was harvested from the femurs, tibias, and pelvis by mechanical disruption using a mortar and pestle in MACS buffer. Cell suspensions were passed through a 70 μm strainer, erythrocytes lysed with ACK buffer, and the resulting cells were stained for flow cytometry.

Muscle immune cell preparation

Mice were perfused with cold PBS containing 2 mM EDTA prior to tissue collection. Tibialis anterior and gastrocnemius-soleus muscles were dissected, trimmed of fat and nerves, and processed for immune-cell isolation using a Percoll gradient. Muscles were minced in IMDM and digested in Collagenase D 1.0 mg/mL, DNase I 30 U/mL in IMDM at 37 °C for 45 min with shaking. Digestion was stopped with I10F, and suspensions were filtered through a 70-μm mesh and pelleted. Cell pellets were resuspended in 40% Percoll–RPMI and overlaid onto 80% Percoll–PBS, then centrifuged at 1,400 × g for 15 min without brake. Leukocytes at the 40%/80% interface were collected, washed with I10F, and stained for flow cytometry.

Immunization

mRNA-LNP:

Cap 1 N1meΨ OVA mRNA was provided by Innovac Therapeutics or purchased from PackGene (Houston, TX). OVA mRNA or dead (non-coding) mRNA LNPs were formulated in lipids at molar ratios of 50:38.5:10:1.5 (ionizable lipid SM-102:cholesterol:DSPC:DMG-PEG2000). LNP size and size distribution, encapsulation efficiency, stability, and endotoxin level were rigorously tested. mLama4 mRNA was provided by Dr. Rober Schreiber (Washington University in St. Louis, MO). For in vivo studies, 50 μl mRNA-LNP containing 10 μg mRNA was injected intramuscularly into the gastrocnemius muscle. Unless indicated otherwise, mRNA-LNP was administered on day 0 and day 7, and the immune responses were measured at day 11.

cDNA vaccine:

Plasmid DNA encoding the full-length OVA was amplified in E.coli DH5α (Invitrogen) and purified using NucleoBond Maxi Plasmid DNA Purification kits (Macherey-Nagel, Bethlehem, PA). Empty pcDNA3.1(+) vector DNA was used as control. DNA vaccination was performed using a Helios gene gun (Bio-Rad, Hercules, CA). Mice were vaccinated with 4 μg of DNA at 3-day intervals (day 0, 3 and 6) for a total of three doses. DNA was delivered to non-overlapping shaved and depilated abdominal areas, with helium discharge pressure set to 400 p.s.i. Immune responses were measured five days after the last gene gun vaccination (day 11).

AddaVax-adjuvanted protein vaccine:

soluble ovalbumin (low endotoxin; Worthington, LS003509) was dissolved in PBS and emulsified 1:1 (v/v) with AddaVax^™ (InvivoGen; vac-adx-10) at 4°C by vortexing for 2 min. Mice were immunized intramuscularly with 50 μl of emulsion containing 10 μg OVA on days 0 and 7, into the same flank.

Necrotic tumor antigens:

freeze-thawed Abelson-mOVA cells were used to standardize antigen quantity without cell proliferation, as previously described¹⁸. Briefly, Abelson-mOVA cells were generated by retroviral transduction of Abl-MuLV-transformed MHC-I TKO BM tumor cell line with a membrane-OVA construct⁵⁷ (Abl-MuLV was a gift from Barry Sleckman, University of Alabama at Birmingham, AL). Cells underwent three rapid freeze-thaw cycles and stored at −20°C until use. Mice were immunized with 3.3 × 10⁵ freeze-thawed Abelson-mOVA cells.

Ex vivo and in vivo OT-I priming assays

Lymph nodes and spleens from CD45.1 OT-I mice were harvested, mechanically dissociated into and passed through 70 μm strainers to make single-cell suspension. Erythrocytes were lysed with ammonium chloride–potassium bicarbonate (ACK) lysis buffer. Cells were depleted of TER-119-, I-A/E-, Ly-6G- and B220-expressing cells by incubation with the biotinylated antibodies for 20 min at 4°C, followed by depletion with MagniSort Streptavidin Negative Selection Beads (Thermo Fisher). Naïve OT-I cells were sorted as B220⁻ CD45.1⁺CD4⁻CD8⁺ Vα2⁺ CD44⁻ CD62L⁻, washed with PBS, and labeled with Cell Trace Violet (CTV) proliferation dyes (Thermo Fisher Scientific).

For ex vivo cross-presentation assays, 2.5 × 10⁴ CTV-labeled OT-I were co-cultured with sorted cDC1, cDC2, or B cells isolated from dLNs or contralateral LNs 2 days after immunization. Co-cultures were performed in a well of U-bottom 96-well plates. After three days, cells were washed, surface-stained with antibodies, and analyzed for CTV dilution and CD44 expression.

For in vivo antigen presentation assays, 5 × 10⁵ CTV-labeled naïve OT-I cells were intravenously transferred into recipient mice. One day later, mice were immunized with indicated antigens. At indicated timepoints, spleens were harvested and erythrocyte lysed with ACK buffer, and CD45.1+ OT-I cells were analyzed for CTV dilution and CD44 expression.

In OT-I proliferation assays, average division number (ADN) was calculated as ∑(fraction of total OT-I cells in division n × n) based on the peak of undivided control without immunization and the peak for each division automatically fit by Flowjo software. The gate boundaries are adjusted to the lowest population between two peaks.

For T cell egress blockade, 1 mg/kg body weight of FTY720 (Sigma, cat. SML0700) was administered by intraperitoneal injection in 150 ul PBS one day after OT-I adaptive transfer.

For blockade of naïve T cell entry to lymphoid organs, splenectomy was performed by WashU Medicine Animal Surgery Core under anesthesia using standard surgical removal of the spleen, followed by closure of peritoneum and skin. Mice were monitored daily for 4 days to ensure recovery. On day 5 after surgery, mice were injected i.p. with 200 ug anti-CD62L (clone MEL-14; Leinco technologies, cat. C2118). Six hours later, mice were adoptively transferred i.v. with CTV-labeled naïve OT-I cells. The following day, mice were immunized with 0.1 ug OVA mRNA-LNP.

CD8⁺ T cell tetramer staining

Spleens were harvested and passed through 70 μm strainers to generate single-cell suspensions. After erythrocyte lysis with ACK lysis buffer, cells were resuspended in MACS buffer. After counting with a ViCell analyzer, 3 × 10⁶ splenocytes were used for staining. APC- and PE-conjugated H-2Kb chicken ova 257-264 SIINFEKL tetramers (NIH Tetramer Core Facility) were added at a concentration of 1:100 in MACS buffer containing 10% Fc Block (2.4G2) and incubated at 37°C for 15 min. Without washing, fluorochrome-conjugated antibodies for surface staining were then added directly and incubated at 4°C for 30 min.

ELISpot assay

ELISpot assay was performed using the Mouse IFN-γ (ALP) ELISpot Plus Kit (Mabtech, Cincinnati, OH) following manufacturer’s instruction. Briefly, 1-2×10⁵ mouse spleen cell suspensions after ACK lysis were incubated in triplicate for 20 hours with or without the presence of 1 μM SIINFEKL peptide (AnaSpec, Fremont, CA). After extensive washes, biotinylated detection antibody was added followed by streptavidin-ALP and insoluble BCIP/NBT-plus substrate. Plates were scanned and analyzed on an ImmunoSpot Reader (CTL, Shanker Heights, OH).

Immunostaining and Microscopy

The contralateral inguinal lymph node was fixed for 6 hours shaking at 4°C in 4% paraformaldehyde (PFA) (Santacruz, sc-281692) that was adjusted to PH 9.0 with triethanolamine. Lymph nodes were washed out of PFA in 1x PBS with 10 U/mL Heparin, embedded in 4% low melt agarose, and sectioned into 200 μm sagittal slices using a LeicaVT1200 vibratome. Sections were blocked in ADAPT-3D blocking buffer⁵⁸ (Leinco, B673) for 1 hour and then stained with primary antibodies against CD11c (Bio-Rad, MCA1369, N418 clone) and F4/80 (BioLegend, 123101, BM8 clone) diluted 1:200 from a stock concentration of 1 mg/mL in ADAPT-3D blocking buffer and left shaking at room temperature overnight. Sections were washed in 1x PBS with 10U/mL Heparin and 0.2% Tween-20, three times for 1 hour each. Sections were stained with secondary antibodies overnight (Jackson ImmunoResearch, Cy3 Goat Anti-Armenian Hamster IgG, 127-165-160; AF647 Donkey anti-Rat IgG, 712-605-153) diluted 1:300 from a stock concentration of 1.5mg/mL in ADAPT-3D blocking buffer after first passing through a 0.22 μm PVDF filter (Millex, SLGVR04NL) and with anti-CD169 (Bio-Rad, MCA947GA, MOMA-1 clone) directly conjugated with CF488 (Biotium, 92253). Sections were washed in 1x PBS with 10 U/mL Heparin and 0.2% Tween-20 three times for 1 hour each. For three sections per lymph node, a tilescan with 9 μm z-stacks was acquired with a 20x lens (air, 0.8NA) on a Leica SP8 confocal microscope. Images were gaussian or median filtered using Imaris v10.1.1 and representative images were exported as a maximum intensity projection.

Tumor experiments

The 1956 tumor cell line expressing membrane-bound ovalbumin (1956-mOVA) was derived from the methylcholanthrene (MCA)-induced fibrosarcoma 1956 tumor (a gift from Robert Schreiber, WashU Medicine, St. Louis, MO), as previously described⁵⁷. The original tumor was generated in a female C57BL/6 mouse, tested for mycoplasma contamination, and banked at low passage⁵⁹. For experiments, tumor cells were thawed from frozen stocks and cultured for 4-6 days in vitro with one intervening passage in RPMI medium supplemented with 2ME, NEAA, glutamine, penicillin/streptomycin, and 10% FBS (R10F).

On the day of injection, tumor cells were harvested by trypsinization, washed three times with PBS, and resuspended at 6.67 × 10⁶ cells/ml. Mice were subcutaneously injected into the shaved flank with 1 × 10⁶ cells. Tumor growth was monitored every 3-5 days with a caliper. Two perpendicular diameters of tumor mass were measured and multiplied to calculate tumor area (mm²). In accordance with IACUC-approved protocol, tumors were not permitted to exceed 20 mm in maximal diameter at any point.

In vivo cytotoxicity assay

In vivo killing assays were performed on mice six weeks after the second OVA mRNA-LNP immunization. Splenocytes from naïve CD45.1+ SJL mice were harvested, ACK lysed, and prepared to single cell suspension. Cells were resuspended in I10F at 2 × 10⁷ cells/ml, and divided into two equal fractions and pulsed with either 1 ug/mL SIINFEKL or 1 ug/mL irrelevant control peptide for 30 mins at 37°C. Cells were then washed twice with PBS and stained at 5 μM for CTV-high or at 0.5 μM for CTV-low for 10 mins at 37°C, and mixed at a 1:1 ratio immediately prior to transfer.

Statistics

Statistical analyses were conducted using GraphPad Prism software version 10. Center values represent the mean, and error bars indicate standard deviations (s.d.) unless otherwise specified. For groups that are not assumed to have equal variances, Welch’s or Brown-Forsythe one-way ANOVA was applied.

Single-Cell Isolation, Library Preparation, and Sequencing

Ova-tetramer-specific splenic cells were isolated from wild-type (WT), Δ32, and Δ1+2+3 mice and washed with 1× phosphate-buffered saline (PBS) containing 0.04% bovine serum albumin (BSA). Prior to fluorescence-activated cell sorting (FACS), cells from each individual mouse were stained with hashtag oligonucleotides (HTOs) to enable multiplexing and improve sample throughput. cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-16 cycles before being cleaned up using SPRIselect beads. Samples were then run on a Bioanalyzer to determine the cDNA concentration. V(D)J target enrichment (TCR) was performed on the full length cDNA. Gene Expression, Enriched TCR and Feature libraries were prepared as recommended by the 10x Genomics Chromium GEM-X Single Cell 5' Reagent Kits User Guide (v3 Chemistry Dual Index) with Feature Barcoding technology for Cell Surface Protein and Immune Receptor Mapping user guide, with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium GEM-X Single Cell 5’ Kit v3, 16 rxns (PN-1000699), Chromium GEM-X Single Cell 5’ Chip Kit (PN-1000698), Chromium Single Cell Mouse TCR Amplification Kits (PN-1000254), Dual Index Kit TT Set A, 96 rxns (PN-1000215), Chromium GEM-X Single Cell 5' Feature Barcode Kit v3, 16 rxns (PN-1000703) and Dual Index Kit TN Set A, 96 rxns (PN-1000250) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer’s protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeqX plus S4 Flow Cell using the XP workflow and a 151x10x10x151 sequencing recipe according to the manufacturer protocol. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression library and 5000 reads/cell for each V(D)J and Feature library. The reads for each sequencing library was then aligned and quantitated with 10x’s CellRanger v9.0.1 against 10x’s standard refdata-gex-mm10-2020-A mouse gene reference and refdata-cellranger-vdj-GRCm38-alts-ensembl-7.0.0 VDJ reference per manufacturer’s protocol.

Single-Cell Gene Expression and TCR Clonotype Analysis

Single-cell gene expression analysis was performed in R (version 4.4.0) using the Seurat package (version 5.3.0)⁶⁰. Hashtag oligonucleotide (HTO) data were first normalized individually for each sample (Supplementary Information Table 2) and demultiplexed using the HTODemux function; only singlet cells were retained for further analysis. Cells with >5% mitochondrial gene expression were excluded, and only those expressing between 200 and 4,000 genes were retained, to remove low-quality cells and potential doublets. Following quality control, data from all samples were merged and normalized. The 3,000 most variable genes were identified, and mitochondrial, ribosomal, and T-cell receptor (TCR) genes were excluded from this list to avoid biases associated with highly abundant or cell type-specific transcripts. The data were then scaled, principal component analysis (PCA) was performed followed by batch correction and data integration using Harmony. Dimensionality reduction of the integrated matrix was carried out using Uniform Manifold Approximation and Projection (UMAP) based on the first 30 principal components. Phenotypic clusters were identified by constructing a k-nearest neighbors (k-NN) graph and applying the Louvain algorithm with a resolution parameter of 0.4.

For TCR repertoire analysis, cell phenotype, sample identity, and mouse ID information were extracted from the integrated metadata for each cell. TCR sequences were successfully annotated for 46,051 cells and used for downstream clonotype analyses. Cells sharing identical CDR3αβ amino acid sequences were defined as belonging to the same TCR clone.

Extended Data

Extended Data Figure 1. Localized mRNA-LNP vaccination can systemically prime CD8 T cells.

a-d, in vivo OT-I proliferation assay at day 0, 2, 3 and 7 after OVA-mRNA immunization. CTV-labeled CD45.1+ naïve OT-I cells were sorted and adoptively transferred to WT mice. 1 day later, mice were i.m. immunized with 10 μg OVA mRNA-LNP. On day 0, 2, 3, or 7 after immunization, spleen, peripheral blood, draining LN, contralateral LN, and mesentery LN were analyzed for OT-I proliferation analysis. a, Schematic of experimental setup for a-c. Data is representative of biologically independent samples per day (0, 2, 3, and 7) from two independent experiments (n=4 per group). b, Representative flow plots of OT-I proliferation. On each day, plots from different tissues are paired from the same mouse. c, Representative histograms of OT-I cells stained with CTV. d, CTV dilution (mean MFI) with different timepoints. (n=4 each.) Two-way ANOVA with multiple comparisons. ns = not significant. e, in vivo OT-I proliferation assay with inhibition of T cell egress from lymphoid tissues. 1 day after OT-I transfer, mice were intraperitoneally (i.p.) injected with PBS or 1 mg/kg body weight FTY720. 6 hours later, mice were injected with OVA mRNA-LNP. (day 2: n=2; day 3: n=4 per group) two-tailed t-test. ***P = 0.0006. f, OT-I cell trafficking to lymphoid organs was blocked by splenectomy and anti-CD62L treatment every two days. 6 hours after the first dose of anti-CD62L, splenectomized mice received CTV-labeled naïve OT-I cells followed by OVA mRNA-LNP immunization (n=6 per genotype.) two-tailed t-test. ****P < 0.0001. Data are represented as mean values ± s.d. of pooled biologically independent samples from two independent experiments.

Extended Data Figure 2. mRNA-LNP systemically primes CD8 T cells in lymphoid tissues.

a, Gating strategy for OT-I in the in vivo OT-I proliferation assay. b, Representative pregating flow plots of OT-I cells from peripheral blood, showing the transient absence of CD8a+ Va2+ CD45.1+ cells specifically on day 2, but not at other timepoints. c, CTV dilution (mean MFI) at the indicated timepoints, following in vivo OT-I proliferation assay at day 2 post OVA-mRNA immunization with different doses. One day after OT-I transfer, mice were immunized i.m. with 0, 0.1, 0.32, 1, 3.2, or 10 μg of OVA mRNA-LNP. Data are represented as mean values of pooled biologically independent samples from one representative experiment from two independent experiments. (n=2 for each condition.) Two-way ANOVA with multiple comparisons. d, e, in vivo OT-I proliferation assay at day 0, 1 and 2 after OVA-mRNA immunization as described in Figure 1 a. d, Representative flow plots showing CD69 expression by OT-I cells at indicated timepoints. e, Frequencies of CD69+ OT-I cells in different tissues across time. Data are represented as mean values of pooled biologically independent samples from two independent experiments. (n=2 for day 0, n=4 for day 1, and n=3 for day 2.)

Extended Data Figure 3. CD8 T cell entry to lymphoid organs is critical for their priming.

a, Representative flow plots showing OT-I proliferation from Fig. 1 f. Gating strategy for cDC1, cDC2, and B cells from LNs after mRNA-LNP immunization. b, c, in vivo OT-I proliferation assay with inhibition of T cell egress from LNs. CD45.1+ CTV+ naïve OT-I cells were adoptively transferred to CD45.2+ wild type mice. 1 day later, mice were intraperitoneally injected with PBS or 1 mg/kg body weight FTY720. 6 hours later, mice were i.m. injected with OVA mRNA-LNP. b, Representative flow plots showing OT-I proliferation on day 2. c Frequencies of proliferating OT-I cells. Data are represented as mean values ± s.d. of pooled biologically independent samples from two independent experiments. (n=4 for each condition.) Two-tailed t-tests. ***P=0.0006, ns=not significant. d-f, OT-I cells in muscle are migrated from lymphoid organs. OT-I cell trafficking to lymphoid organs was blocked by splenectomy and anti-CD62L treatment every two days. 6 hours after the first dose of anti-CD62L, splenectomized mice received CTV-labeled naïve OT-I cells followed by OVA mRNA-LNP immunization. Data are represented as mean values ± s.d. of pooled biologically independent samples from three independent experiments. (n=6 for each condition.) Two-way ANOVA. *P=0.0486, ns=not significant.

Extended Data Figure 4. Both cDC1 and cDC2 prime CD8 T cells after mRNA vaccination.

a, Gating strategy for cDC1, cDC2, and B cells from LNs after mRNA-LNP immunization. b, Gating strategy for tetramer+ CD8 T cells from spleen. c, ELISpot results from cDNA vaccination experiment of Fig. 2 d, e. P**=0.0037, P***=0.0008, ns=0.8135. d, e, WT and Δ32 mice were injected i.m. with 10 μg OVA adjuvanted with AddaVax on day 0 and 7. Spleens were stained for SIINFEKL-K^b tetramer+ CD8 T cells and CD44/CD62L expression on day 11. Data represents pooled biologically independent samples from three independent experiments. (n=15 WT. n=9 Δ32.) One-way ANOVA with Tukey’s multiple comparisons. P**=0.0081. f, IFNg ELISpot results from Fig. 3 e, f. (n=7 Cre-, n=8 Cre+.) One-way ANOVA with Tukey’s multiple comparisons. g, SIINFEKL-K^b-PE MFI from tetramer+ CD8 T cells from the experiment shown in Fig. 2 f, g. (n=9 WT, n=11 Δ32, and n=11 Δ1+2+3.) One-way ANOVA with Tukey’s multiple comparisons. h, SIINFEKL-K^b tetramer+ CD8 T cells on day 7 after mLama4 mRNA-LPX vaccination in WT, Δ32, and Δ1+2+3 mice. (unimmunized: n=4; immunized: n=8 WT, n=7 Δ32, and n=7 Δ1+2+3.) One-way ANOVA with Tukey’s multiple comparisons. P***=0.0003. Data represented as mean values ± s.d. and pooled biologically independent samples from two independent experiments. ns = not significant.

Extended Data Figure 5. cDCs are required to prime CD8 T cells.

a, Verification of WT, Δ32, and Δ1+2+3 mice were injected i.m. with OVA mRNA-LNP. 7 days later, spleens were analyzed for the presence of SIINFEKL-Kb tetramer+ CD8 T cells. One-way ANOVA b, c, Verification of splenic cDC depletion after DT treatment in CD11c-DTR mice from the experiments shown in Figure 2 h-i. One-way ANOVA. d, Verification of splenic cDC depletion after DT treatment in CD11c-DTR bone marrow chimera mice from the experiments shown in Figure 2 j. Two-tailed t-test. e, f, Quantification of macrophage population after DT treatment in CD11c-DTR mice from the experiments shown in Figure 2 h-i. One-way ANOVA. g, h, Quantification of inflammatory monocyte population after DT treatment in CD11c-DTR bone marrow chimeras from the experiments shown in Figure 2 j. Two-tailed t-test. i, Representative images of inguinal LN with or without DT treatment in CD11c-DTR BM chimeras from two independent experiments. CD11c is shown in turquoise, CD169 in red, F4/80 in green, and DAPI in blue. j-l, WT and Wdfy4^−/− mice were injected with OVA mRNA-LNP on day 0 and 7. Spleens were stained for the presence of SIINFEKL-K^b tetramer+ CD8 T cells and their CD44 and CD62L surface expression on day 11. (n=6 for WT, n=7 for Wdfy4^−/−.) j, Representative flow plots of CD8 T cells. k, Quantification of tetramer+ CD44+ CD62L+ CD8 T cells as a percentage of CD8 T cells. Two-tailed t-test. l, IFNg ELISpot results. Two-tailed t-test. Data represented as mean values ± s.d. and pooled biologically independent samples from two independent experiments P**** < 0.0001, ns = not significant.

Extended Data Figure 6. cDCs and BM progenitors were exclusively derived from donor.

a, Gating strategy for splenic cDC1 and cDC2 to examine BM chimerism, from a representative MHC-I TKO → SJL BM chimera. b, Quantification of splenic cDC chimerism. c, Gating strategy for CDP, MDP, pre-cDC1, and pre-cDC2 populations to examine bone marrow chimerism. (n=6 each.) d, Quantification of BM progenitor chimerism. (n=6 each.) Data represented as mean values ± s.d. and pooled biologically independent samples from two independent experiments.

Extended Data Figure 7. Type I IFN signaling mediates cross-dressing following mRNA vaccines.

a-d, Tetramer staining of CD8 T cells in peripheral blood (a, b) and draining LN (c, d) from IFNAR1 blockade experiment from Figure 5. b, d, One-way ANOVA with Tukey’s multiple comparisons. P****<0.0001, P**=0.0061. e, f, Itgax-Cre⁺ β2m^fl/fl mice were immunized with OVA mRNA-LNP, with or without 2 mg anti-IFNAR1 treatment one day before immunization. 4 days after immunization, splenocytes were analyzed for H2-K^b expression on cDCs. (n=4 per genotype.) Two-tailed t-test. Data are represented as mean values ± s.d. from pooled biologically independent samples from two independent experiments.

Extended Data Figure 8. cDCs acquire non-self MHC-I from non-hematopoietic cells.

a, Acquisition of H2-K^b expression in CD45.2+ splenic cDCs from BM chimera of Figure 3 h, i. Data shown is representative flow plots from two independent experiments. b, Gating strategy for splenic cDC1 and cDC2. c, d, BM chimerism of B6 and BALB/c allogeneic BM chimeras from Figure 4. c, Representative flow plots of lineage- cKit^high BM progenitors. d, Quantification of lineage- cKit^high BM progenitor chimerism. e, f, Acquisition of H2-K^b expression in Balb/c+ splenic cDCs from B6 and BALB/c allogeneic BM chimera shown in Figure 4. Data shown is representative flow plots from two independent experiments. Two-tailed t-test. P*=0.033. g, h Tetramer response from B6 and BALB/c allogeneic BM chimeras from Figure 4. (For unimmunized control: n = 1-2 per condition. For OVA mRNA-LNP, n = 7 for BALB/c→B6, n = 3 for BALB/c→BALB/c, n = 6 for B6→B6, and n=6 for B6→BALB/c mice.) Data are represented as mean values ± s.d. from pooled biologically independent samples from two independent experiments.

Extended Data Figure 9. CD8 T cell memory and functions.

a-c, Memory SIINFEKL-Kb tetramer+ CD8 T cells after OVA mRNA–LNP vaccination. WT, Δ32, and Δ1+2+3 mice were immunized on days 0 and 7 with OVA mRNA–LNP, and peripheral blood was analyzed 6 weeks after the second dose. a, Frequencies of SIINFEKL-Kb tetramer+ CD8 T cells. One-way ANOVA with Tukey’s multiple comparisons test. b, Representative flow plots of tetramer+ CD8 T cells showing CD127 and KLRG1 expression. c, Quantification of CD127+ cells among tetramer+ CD8 T cells. (n=8 WT, n=7 Δ32, and n=6 Δ1+2+3.) One-way ANOVA. *P (left) = 0.0125, *P (right) = 0.0215. d-g, In vivo cytotoxicity assay. Six weeks after the second dose of OVA mRNA-LNP, mice received a 1:1 mixture of irrelevant peptide-loaded (CTV-low) or SIINFEKL-loaded (CTV-high) CD45.1+ splenocytes. d, Representative CTV histograms of transferred target cells. e, Killing efficiency normalized to WT. (n=10 WT, n=13 Δ32, and n=9 Δ1+2+3.) One-way ANOVA with Tukey’s multiple comparisons test. *P = 0.0474. f, g, Frequencies of SIINFEKL-Kb tetramer+ CD8 T cells in spleen (f) and LNs (g) (n=8 WT, n=9 Δ32, and n=9 Δ1+2+3.) ****P < 0.0001, **P = 0.0014, *P = 0.0429. Data are represented as mean values ± s.d. from pooled biologically independent samples from two independent experiments. ns = not significant.

Extended Data Figure 10. Identification of clusters in single cell analysis of CD8 T cells.

Heatmap displaying pseudobulk expression profiles of the top 50 differentially expressed genes across experimental conditions, stratified by cell cluster. Gene expression was aggregated at the cluster level per condition to highlight transcriptional shifts in response to immunization.

Data availability

The single-cell RNA- and TCR-sequencing data underlying Fig. 6 and Extended Data Fig. 7 is openly available in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) with the accession number GSE296093. All data in this study are available in the published article and its supplemental materials.