Research ArticleMALARIA

Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against rodent malaria

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Science Immunology  26 Jun 2020:
Vol. 5, Issue 48, eaaz8035
DOI: 10.1126/sciimmunol.aaz8035

Defending the liver

The liver is an important site of replication for Plasmodium parasites, and therefore a key goal in vaccination against malaria is to induce robust antiparasitic immunity in the liver. Using Plasmodium berghei as a model to study malaria in mice, Holz et al. have developed a glycolipid-peptide conjugate vaccine that induced robust T cell responses in the liver and was able to protect mice when challenged with P. berghei. Inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) that activates natural killer T (NKT) cells was vital to promoting antiparasitic immunity in the liver. The authors propose that agonists that activate NKT cells could be useful in priming immune responses in the liver in the context of malaria and in other hepatotropic diseases.


Liver resident-memory CD8+ T cells (TRM cells) can kill liver-stage Plasmodium-infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver TRM cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I–restricted peptide epitope (to stimulate Plasmodium-specific CD8+ T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8+ T cells expressing canonical markers of liver TRM cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver TRM cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against Plasmodium berghei sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver TRM cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver TRM cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections.


Malaria is a highly prevalent parasitic disease, accounting for about 216 million infections and 445,000 deaths per annum (1). It is caused by Plasmodium spp. and is transmitted via female Anopheles mosquitoes. After a bite from an infected mosquito, immature parasites (sporozoites) injected into the skin circulate via the blood to the liver where they infect hepatocytes. Over about 1 week in humans, or 2 days in mice, sporozoites mature and replicate within hepatocytes and are released into the blood as merozoites, which infect red blood cells and cause disease [reviewed in (2)]. Vaccination with radiation-attenuated sporozoites (RAS) was demonstrated 30 years ago to elicit sterile protection, in part, by targeting the liver stage of the parasite life cycle (3). This protection is mediated by CD8+ T cells (46), and large numbers of memory CD8+ T cells are required (7, 8).

Memory CD8+ T cells can be separated into central memory (TCM) and effector memory (TEM), which recirculate, and tissue-resident memory (TRM), which remains permanently within tissues (9). Liver TRM cells reside in the vasculature (liver sinusoids), scanning underlying hepatocytes for infection (10, 11). Recently, protection induced by RAS vaccination was shown to be dependent on liver TRM cells (10, 12). As a proof of concept, we developed a complex “prime-and-trap” (P&T) vaccination approach that was able to induce liver CD8+ TRM cells and was superior to the current gold standard vaccine, RAS, at protecting against malaria (10). However, a requirement for three different components somewhat limits the versatility of this vaccination approach.

A malaria vaccine that is stable and easy to manufacture and administer is currently lacking [reviewed in (2)]. Given the requirement for liver TRM cells for protection, any new vaccine must be capable of generating these cells. To address such needs, we examined the utility of a glycolipid-peptide vaccine that uses help from type 1 natural killer T (NKT) cells to generate potent CD8+ T cell responses (1316). To use NKT cell help in vaccination, we conjugated our CD8+ T cell antigenic peptide epitope to an NKT cell agonist α-galactosylceramide (α-GalCer) to facilitate uptake of both components by the same antigen-presenting cell (APC). To facilitate conjugation, an N → O acyl-migrated isomer of α-GalCer was used (MaGC), with the exposed nitrogen used to attach peptide via a linker containing a cathepsin-sensitive valine-citrulline–p-amino-benzyl (VC-PAB) carbamate that immolates after cleavage once the conjugate is acquired by APC. A reverse O → N acyl migration is then favored within MaGC, forming α-GalCer, which can be presented via CD1d to NKT cells. The released peptide is designed with an N-terminal proteasomal cleavage sequence, Phe-Phe-Arg-Lys (FFRK), that promotes release of the peptide component from the linker to favor processing and presentation via major histocompatibility complex (MHC) molecules to T cells. NKT cells respond rapidly to glycolipid antigens including α-GalCer, which are presented in the context of CD1d molecules on APC [reviewed in (17)]. Recognition leads to “licensing” of the APC through CD40L interactions and soluble factors (13, 14) and enhances responses by CD8+ T cells (13), including those induced by RAS (18). Our earlier studies have shown that glycolipid-peptide conjugates are capable of inducing strong circulating CD8+ T cell responses effective against tumors (19) and influenza (20), but whether liver TRM cells were induced was untested. Because antigen presentation and inflammation within the liver are key drivers of TRM cell generation (10, 11, 2123) and NKT cells are a prominent population that migrate within the liver sinusoids (24), we speculated that their response to our glycolipid-peptide vaccine might drive TRM formation. Here, we describe the development of a malaria-specific glycolipid-peptide conjugate vaccine that is able to generate large numbers of CD8+ TRM cells in the liver and protect against pre-erythrocytic Plasmodium infection, preventing malaria.


A glycolipid-peptide vaccine induces OVA-specific liver CD8+ TRM cells

To assess whether our glycolipid-peptide vaccination approach could induce liver TRM cells, C57BL/6 (B6) mice were adoptively transferred with 40,000 naïve OT-I transgenic T cells [specific for SIINFEKL, an H-2Kb–restricted epitope of ovalbumin (OVA)] and then vaccinated intravenously with V.FFRK.OVALP (Fig. 1A; structure shown in fig. S1), a glycolipid-peptide conjugate of a “long peptide (LP)” containing the I-Ab and H-2Kb epitopes of OVA attached to MaGC. Control groups of mice were vaccinated intravenously with 600 plaque-forming units (PFU) of a modified influenza A virus, PR8-OVA, which expresses SIINFEKL (25), or with unconjugated α-GalCer and the LP. Livers were harvested on days 21 and 60 to examine liver TRM cell formation as assessed by staining for markers CD69 and CD62L (Fig. 1B and fig. S2). Large numbers of OT-I liver TRM cells (~106 CD69+CD62L cells) were induced by V.FFRK.OVALP (Fig. 1C). These cells were detected as soon as day 21 and persisted for more than 60 days (Fig. 1, C, D, F, and G). These CD69+CD62L OT-I cells displayed a liver TRM cell phenotype, with high expression of CXCR6, CD49a, and CD101 and low expression of KLRG1 and CX3CR1 (Fig. 1B). In contrast, neither PR8-OVA nor the unconjugated α-GalCer with peptide efficiently induced liver TRM cells (Fig. 1, C and F). Increasing the dose of PR8-OVA to 106 PFU yielded similar findings (fig S3). While the unconjugated α-GalCer and peptide was also poor at stimulating memory T cells in the spleen, both PR8-OVA and V.FFRK.OVALP induced splenic OT-I effector and central memory (TEM and TCM) cells (Fig. 1, E and H). Small numbers of splenic T cells of a TRM cell phenotype were also induced by the glycolipid-peptide conjugate, but not by other vaccines (Fig. 1, E and H). From this analysis, we conclude that while both PR8-OVA and V.FFRK.OVALP induce circulating memory T cells, only V.FFRK.OVALP promoted efficient differentiation into liver TRM cells. Furthermore, chemical linkage between the two vaccine components is required to achieve efficient priming.

Fig. 1 V.FFRK.OVALP generates large numbers of liver TRM cells.

(A) Ly5.1+ OT-I cells (40,000) were transferred into recipient B6 mice. One day later, the recipient mice were treated intravenously with 600 PFU PR8-OVA, 0.135 nmol of α-GalCer and OVA LP [αGC + OVALP], or 0.135 nmol of a conjugate vaccine containing OVALP [V.FFRK.OVALP] (see fig. S1). Organs were harvested from recipient mice on days 21 and 60, and memory T cells were assessed by flow cytometry (see fig. S2). (B) Top: Expression of CD62L and CD69 by OT-I.Ly5.1 cells (CD8+Ly5.1+). Bottom: Phenotype of TRM cells (CD8+ Ly5.1+ CD44+ CD69+ CD62Llow) and TEM cells (CD8+ Ly5.1+ CD44+ CD69 CD62Llow) in the liver on day 21 after vaccination with V.FFRK.OVALP. (C and F) Number of liver TRM cells on days 21 (C) and 60 (F). (D, E, G, and H) Number of liver (D and G) and spleen (E and H) TRM, TEM, and TCM (CD8+ Ly5.1+ CD44+ CD69 CD62L+) cells on days 21 (D and E) and 60 (G and H). Results in (B) to (H) are from one to two independent experiments with five mice per group. Data displayed show means ± SEM or individual mice. Groups in (C) and (F) were compared by one-way ANOVA with Tukey’s multiple comparison posttest. ****P < 0.0001.

Modifications to the acyl chain length limit NKT cell activation and liver TRM cell formation

To explore the relationship between NKT cell activation, liver inflammation, and liver TRM cell induction, a suite of OVALP-based conjugates was synthesized in which the length of the acyl chain was incrementally reduced from the natural length of 26 carbons down to 0 carbons (C26 to C0). Decreasing the acyl chain length was envisaged to reduce CD1d binding and therefore reduce NKT cell stimulation and liver inflammation. B6 mice were vaccinated with these conjugates and then assessed for serum alanine aminotransferase (ALT) levels, NKT cell activation, and liver TRM cell formation (Fig. 2 and movie S1). NKT cells that patrol the liver sinusoids (24) showed some changes in migration behavior after vaccination (movie S1), indicating their response in the liver. Examination of serum ALT 18 hours after vaccination as a measure of liver inflammation revealed normal serum levels for chain lengths below C22 and a gradual rise in ALT levels with increasing length (Fig. 2A). ALT levels mirrored induction of NKT cell proliferation as measured by the number of NKT cells in the spleen and liver (Fig. 2, C and D) and with down-regulation of CD69 on NKT cells, a marker of activation (Fig. 2, E and F). NK1.1 expression, which also down-regulates upon NKT cell activation, was reduced for all compounds, indicating some level of stimulation even for shorter chain conjugates (Fig. 2, G and H). The C26 vaccine, which contains the same number of carbon atoms in the fatty acid chain as α-GalCer, triggered lower NKT cell activation (Fig. 2, C, E, F, and H) compared with direct injection of α-GalCer, suggesting incomplete O → N acyl migration or loading of α-GalCer onto CD1d molecules in the C26 vaccine. Examination of liver TRM cell formation at 3 weeks after vaccination (Fig. 2B) revealed a similar pattern of variation to that seen for serum ALT levels (Fig. 2A) and NKT cell activation (Fig. 2, D to G), with TRM cell responses gradually waning with chain length reduction until a marked loss at C8.

Fig. 2 Acyl chain length affects liver NKT cell activation and TRM cell formation.

B6 mice were injected with 50,000 Ly5.1+ OT-I cells and one day later vaccinated intravenously with either PBS, 0.135 nmol of α-GalCer, or 0.135 nmol of conjugate vaccines containing the OVALP and MaGC with acyl chains of varying lengths [V.FFRK.OVALP (C26 to C0) referenced simply by their acyl chain length (C26 to C0) on the x axis]. (A) Serum ALT levels 18 hours after vaccination. (B) Number of liver TRM cells on day 21. (C and D) Number of NKT cells in the spleen (C) and liver (D) 3 days after vaccination. (E and F) CD69 expression on NKT cells in the spleen (E) and liver (F) on day 3. (G and H) Percentage of NKT cells in the spleen (G) and liver (H) expressing NK1.1. Results from two independent experiments using at least five mice per group. Groups in (A) and (C) to (H) were compared with the PBS group, and C26 group was compared with α-GalCer group by one-way ANOVA with Sidaks’s multiple comparison posttest. Groups in (B) were compared with the C26 group by one-way ANOVA with Sidaks’s multiple comparison posttest. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Nonsignificant differences are not shown.

Together, these findings suggest that the strength of NKT cell activation can be controlled by modification of the acyl chain length and mirrors the number of TRM cells generated in the liver. Collectively, therefore, modification of the acyl chain length may be used to optimize liver TRM cell induction while minimizing liver inflammation. Because the full-length acyl chain C26 yielded the maximum number of liver TRM cells, we used this length in all subsequent vaccines.

A glycolipid-peptide vaccine induces protective Plasmodium-specific liver CD8+ TRM cells

To determine whether our vaccine could generate liver TRM cells against a liver pathogen, i.e., Plasmodium berghei ANKA (PbA) (which causes malaria), we generated a conjugate (termed V.FFRK.NVYSP) containing a short peptide (SP) epitope, NVYDFNLL, recognized by Plasmodium-specific PbT-I T cell receptor (TCR) transgenic CD8+ T cells, linked to MaGC (fig. S1). Note that NVYDFNLL was identified by a combinatorial peptide library approach (26) and mimicked the authentic PbA epitope recognized by PbT-I cells, which had yet to be identified.

B6 mice were adoptively transferred with 50,000 naïve PbT-I.GFP (green fluorescent protein) cells and then vaccinated intravenously with either the glycolipid-peptide conjugate or an established (positive control) P&T vaccine (10). The P&T regime consisted of two steps: intravenous vaccination with CpG oligonucleotide plus a Clec9A-specific monoclonal antibody covalently linked to NVYDFNLL and then, the following day, intravenous inoculation with a nonreplicating recombinant adeno-associated virus (rAAV) encoding for hepatocyte-specific expression of NVYDFNLL. Analyses on day 35 revealed that PbT-I liver TRM cells were generated in response to both P&T and V.FFRK.NVYSP, with slightly lower numbers in the V.FFRK.NVYSP group (Fig. 3, A and C). A similar trend was observed for TEM cell numbers in the liver and spleen, namely, slightly lower numbers in the V.FFRK.NVYSP group (Fig. 3, C and D). Liver TRM cells generated through vaccination with V.FFRK.NVYSP expressed high levels of CXCR6, CD49a, and CD101 (Fig. 3B) and patrolled the liver sinusoids (movie S2); phenotypes identical to those described for P&T-generated TRM cells (10). The numbers of liver TRM cells generated in the malaria system (Fig. 3) were approximately 10-fold lower than those elicited in the OVA system (Fig. 1).

Fig. 3 Vaccination with V.FFRK.NVYSP protects against malaria.

B6 mice were transferred with 50,000 PbT-I.GFP cells and 1 day later vaccinated intravenously with anti–Clec9a-NVY + CpG (and 1 day later AAV-NVY) (P&T) or α-GalCer (αGC) or 0.135 nmol of a conjugate containing NVYSP (V.FFRK.NVYSP). Organs were harvested day 35 after vaccination, and T cell memory was assessed. (A) Number of liver TRM cells (CD8+GFP+CD44+CD69+CD62LlowKLRG1). (B) Phenotype of liver TRM (red lines) and TEM cells (blue lines) after vaccination with V.FFRK.NVYSP. (C and D) The number of PbT-I TRM, TEM cells (CD44+ CD69 CD62Llow), and TCM (CD44+ CD69 CD62L+) cells in the liver (C) and spleen (D) on day 35 after vaccination. (E and F) Additional mice were challenged with 200 P. berghei sporozoites on day 42. (E) Parasitemia day 7 after challenge. (F) Percentage of mice protected (white) or not (black) after challenge. Absolute numbers are shown above bars. Results from two to three independent experiments using at least four mice per group for each, except for the naïve group in (A) and (C) with only three mice. Data displayed show means ± SEM or individual mice. Groups in (A) and (E) were compared by one-way ANOVA with Tukey’s multiple comparison posttest. Groups in (F) were compared using Fisher’s exact test. ****P < 0.0001.

To assess the conjugate vaccine V.FFRK.NVYSP for its capacity to confer sterile protection against sporozoite challenge, similar groups of mice as those analyzed above were challenged with 200 PbA sporozoites, equivalent to the parasite load in 1 to 2 mosquito bites (27). Because sporozoites infect hepatocytes for 2 days and are then released into the blood as merozoites (28), liver-stage protection can be measured by examining the blood for parasitemia 6 to 13 days after infection (Fig. 3, E and F). As previously reported (10), nearly all mice (13 of 14 mice) vaccinated by our established P&T vaccine were protected from infection. A significant proportion (9 of 12) was also protected after vaccination with V.FFRK.NVYSP, whereas no mice were protected after receiving α-GalCer alone (Fig. 3F). These data show that glycolipid-peptide vaccine conjugates can protect against sporozoite infection.

Prime and boost vaccination using the glycolipid-peptide conjugate enhances protection

Because one immunization with V.FFRK.NVYSP failed to protect all mice, we investigated whether a booster immunization would increase liver TRM cell numbers and improve protection (Fig. 4A). B6 mice were adoptively transferred with 50,000 PbT-I cells and then primed intravenously with V.FFRK.NVYSP. After 21 to 30 days, these mice were boosted with V.FFRK.NVYSP, and memory T cells were examined after day 50 (Fig. 4, B to D, column 5). Homologous boosting with V.FFRK.NVYSP induced an increase in PbT-I liver TRM cell numbers and increased memory T cells in the spleen compared with groups of mice receiving only a single dose of vaccine either at the priming or booster stage (Fig. 4, B to D, columns 2 and 3). It should be noted that mice primed at the booster stage 21 to 30 days after naïve PbT-I transfer (Fig. 4, C and D, column 3) showed much lower memory responses than those primed 1 day after PbT-I transfer either at the priming stage or booster stage (Fig. 4, C and D, columns 2 and 4, respectively), possibly because of poor survival of naïve PbT-I cells between days −1 and 30 before vaccination of this group. Irrespective of when PbT-I cells were transferred, however, the prime-boost regimen generated significantly higher TRM cell numbers compared with single priming.

Fig. 4 V.FFRK.NVYSP can prime-boost.

(A) PbT-I.GFP cells were transferred into B6 or CD1d−/− mice. CD1d−/− mice were then vaccinated intravenously with V.FFRK.NVYSP days 0 and 30 (group 1). B6 mice were vaccinated with V.FFRK.NVYSP on day 0 (group 2), day 30 (group 3), or on both days (group 5). Group 4 B6 mice received PbT-I.GFP cells on day 29 and were vaccinated on day 30 with V.FFRK.NVYSP. Additional mice were vaccinated with anti–Clec9a-NVY + CpG on day 0 alone (group 7) or also boosted on day 30 with V.FFRK.NVYSP (group 6). Memory T cells were assessed on days 50 to 60. (B) Number of liver PbT-I TRM cells. (C and D) Number of PbT-I TRM, TEM, and TCM cells in the liver (C) and spleen (D). Similarly vaccinated mice were challenged with 200 P. berghei sporozoites on days 51 to 73, and survivors were rechallenged with 3000 sporozoites on day 93. (E) Parasitemia on day 7 after initial challenge. (F) Percentage protected (white) or not (black) against 200 sporozoites or protected against 200 and 3000 sporozoites (gray). Absolute numbers are shown above bars. Results from >1 independent experiment with >3 mice per group. Data shown are means ± SEM or individual mice. (B and E) One-way ANOVA with Tukey’s multiple comparison posttest. (F) Fisher’s exact test. *P < 0.05 and ****P < 0.0001.

As an alternative, a heterologous prime-boost vaccination method was also used where mice were vaccinated with CpG + anti–Clec9A-NVY (without the virus used in P&T) and then boosted with V.FFRK.NVYSP or left unboosted (Fig. 4, B to D, columns 6 and 7, respectively). This also induced a substantial increase in TRM cells, showing that V.FFRK.NVYSP was also very effective at heterologous boosting. The boosting potential of the conjugate vaccines was far more pronounced in the heterologous setting, suggesting some residual down-regulation of NKT cell function even after 30 days or induction of partially neutralizing antibodies in the homologous setting. Alternatively, circulating cells induced by the Clec9A stimulus may be more amenable to conversion to liver TRM cells upon heterologous boosting by the conjugate. The dependence on help from NKT cells for expanding PbT-I responses with V.FFRK.NVYSP was demonstrated by poor responses in CD1d−/− mice (Fig. 4, B to D, column 1).

To explore protection induced by these prime-boost regimens, mice were initially challenged with 200 P. berghei sporozoites (Fig. 4, E and F). Both prime-boost regimens (Fig. 4, E and F, columns 6 and 7) induced sterile protection to this robust challenge in all mice. Surviving mice were then rechallenged on day 93 with a very high dose of 3000 sporozoites (Fig. 4F), revealing protection of more than half of the mice from each prime-boost group. Together, these data indicate that V.FFRK.NVYSP can be used in prime-boost regimens and that this vaccine induces large numbers of liver TRM cells that efficiently protect against sporozoite challenge.

Vaccination with conjugate vaccines containing epitope-flanking sequences improves liver TRM cell generation

To test whether liver TRM formation induced by the minimal PbT-I T cell epitope NVYDFNLL could be enhanced, we added additional sequences to the N and C termini of the peptide, which might prevent its degradation and/or improve epitope processing. Because a longer peptide containing an AAA spacer and four flanking amino acid residues at either termini from the protein antigen sequence (AAA-HSLS-NVYDFNLL-LERD) generated large numbers of liver TRM after P&T vaccination (10), two new glycolipid conjugate vaccines containing this longer peptide variant were generated, one with an additional -FFRK- proteasomal cleavage sequence after the AAA spacer (vaccines termed V.NVYLP and V.FFRK.NVYLP, respectively) (fig. S1). Groups of mice received 50,000 PbT-I T cells and were vaccinated the following day with α-GalCer alone, unconjugated vaccine components α-GalCer and NVYLP, or conjugate vaccines V.FFRK.NVYSP, V.FFRK.NVYLP, or V.NVYLP. This also allowed additional assessment of the requirement for the FFRK sequence. Analysis at day 21 revealed that V.FFRK.NVYLP was most effective at inducing liver TRM cells (Fig. 5, A and B, column 5) and splenic TEM and TRM cells (Fig. 5C). The conjugate vaccine lacking FFRK generated the lowest liver TRM cell numbers. Consistent with this observation, early analysis (day 3 after treatment) revealed that this vaccine generated the smallest increase in hepatic and splenic NKT cell numbers (fig. S4, A and B, column 4), the poorest activation of these cells (fig. S4, C to F), and failed to increase serum ALT levels (fig. S4G).

Fig. 5 Conjugate vaccines containing peptide flanking residues generate large numbers of liver TRM cells.

B6 mice were transferred with 50,000 PbT-I.GFP cells and, 1 day later, vaccinated intravenously with α-GalCer alone (αGC), α-GalCer + NVYLP, or conjugates V.FFRK.NVYSP, V.FFRK.NVYLP, or the latter without FFRK (V.NVYLP). Organs were harvested on days 21 to 35 after vaccination, and memory T cells were assessed. Similar groups were challenged with 200 P. berghei sporozoites on day 42, and parasitemia was assessed. (A) Number of liver TRM cells. (B and C) The number of PbT-I TRM, TEM cells, and TCM cells in the liver (B) and spleen (C). (D) Parasitemia on day 7 after challenge. (E) Percentage of mice protected (white) or not (black) after challenge. Absolute numbers are shown above bars. Results from two to three independent experiments with >3 mice per group. Data shown are means ± SEM or individual mice. (A and D) One-way ANOVA with Tukey’s multiple comparison posttest. (E) Fisher’s exact test. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

To determine the level of protection these vaccines provided, vaccinated mice were challenged with 200 P. berghei sporozoites and blood-stage parasitemia monitored (Fig. 5, D and E). Complete protection from sporozoite challenge was observed in all mice treated with V.FFRK.NVYSP or V.FFRK.NVYLP and in about 50% of mice treated with V.NVYLP, a result reflective of the liver TRM cell numbers generated through vaccination (Fig. 5A). Combined, these data demonstrate the advantage of using C- and N-terminally extended peptides with an additional FFRK N-terminal cleavage sequence in NVYDFNLL conjugate vaccines for generation of maximum numbers of liver TRM and protection.

Vaccination with conjugate vaccines synthesized by oxime ligation improves liver TRM cell generation

Both here (Figs. 1 to 5) and elsewhere (20), we have successfully used strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry to manufacture conjugate vaccines. Given that the chemical motif that results from SPAAC conjugation has a relatively large chemical size, we investigated oxime ligation as an alternative conjugation chemistry with a smaller chemical footprint. The NVYDFNLL short peptide was therefore N-terminally modified with aminooxyacetic acid for oxime ligation and conjugated with MaGC to give VOx.FFRK.NVYSP (fig. S1). To confirm that the linker could be accessed enzymatically, both VOx.FFRK.NVYSP and V.FFRK.NVYSP vaccines were incubated with cathepsin B (fig. S5), an enzyme expressed by dendritic cells (DCs) that cleaves the VC-PAB linker (29). MaGC was only detectable for the oxime vaccine (fig. S5, A and B), suggesting that this vaccine may be more efficiently processed.

To test the efficacy of the oxime vaccine, B6 mice adoptively transferred with 50,000 PbT-I T cells were vaccinated intravenously with either SPAAC or oxime versions of the same vaccine. Mice vaccinated with VOx.FFRK.NVYSP generated significantly higher numbers of liver TRM cells (Fig. 6A), whereas TCM and TEM cell numbers were similar between groups (Fig. 6, B and C). Vaccinated mice challenged at day 35 with 200 PbA sporozoites all showed sterile immunity (Fig. 6, D and E). An additional group of mice that survived challenge with 200 sporozoites was rechallenged with 3000 sporozoites on day 58, and again all vaccinated mice survived (Fig. 6, F and G). These results show that the oxime version of the NVYSP vaccine induced greater numbers of liver TRM cells, but both oxime and SPAAC vaccines induced sufficient TRM cells to achieve sterile protection. The greater number of liver TRM cells induced by the oxime vaccine is important, however, because it will likely extend the period of protection as liver TRM numbers wane with time.

Fig. 6 Conjugates using oxime chemistry elicit sterile immunity against Plasmodium.

B6 mice were transferred with 50,000 PbT-I.GFP cells and, one day later, vaccinated intravenously with V.FFRK.NVYSP linked by SPAAC or oxime chemistry. Organs were harvested on day 21, and memory T cells were assessed. Similar groups of mice were challenged with 200 P. berghei sporozoites on day 35, and parasitemia was assessed. Some mice were rechallenged with 3000 P. berghei sporozoites on day 58, and parasitemia was assessed. (A) Number of liver TRM cells on day 21. (B to C) Number of PbT-I TRM, TEM, and TCM cells in the liver (B) and spleen (C) on day 21 after vaccination. (D) Parasitemia on day 7 after 200 sporozoite challenge. (E) Percentage of mice protected (white) or not (black) after 200 sporozoite challenge. Absolute numbers are shown above bars. (F) Parasitemia day 7 after 3000 sporozoite challenge. (G) Percentage mice protected ((grey) or not (black) after 3000 sporozoite challenge. Absolute numbers shown above the bars. Results from one (F and G) or two independent experiments using at least four mice per group. Data shown are means ± SEM or individual mice. (A, D, and F) One-way ANOVA with Tukey’s multiple comparison posttest. (E and G) Fisher’s exact test. ****P < 0.0001.

Conjugate vaccines generate long-lived endogenous TRM cells

To this point, all experiments used B6 mice seeded with transgenic T cells. This questioned whether conjugate vaccines could induce endogenously derived liver TRM cells capable of protection. As previously mentioned, in our initial experiments, we used the antigenic mimic epitope NVY to stimulate PbT-I T cells as we had not identified their cognate malaria antigen. However, more recently, we identified this antigen as NVFDFNNL (NVF), a Kb-binding epitope from the 60S ribosomal protein L6 (RPL6; PBANKA_1351900) (30). This epitope was incorporated into an optimized conjugate vaccine (VOx.FFRK.NVFLP; fig. S1) and used to examine the endogenous T cell response and protection (Fig. 7). B6 mice were vaccinated with VOx.FFRK.NVFLP and, 1 month later, examined for the generation of NVF-specific CD8+ T cells using Kb-NVF tetramers (Fig. 7A, left). This revealed large numbers of NVF-specific TRM cells in the liver and substantial memory T cell populations in the spleen (Fig. 7A, right). This response was partially dependent on CD40 (fig. S6), consistent with NKT cell licensing of DCs. To explore the protective capacity of endogenous NVF-specific T cells, mice were initially challenged on day 42 with 200 PbA sporozoites, and then surviving mice were rechallenged 1 month later with 3000 sporozoites (Fig. 7B). All mice survived the low-dose challenge, and 80% survived the high-dose challenge, indicating strong efficacy. When used in a prime-boost setting (Fig. 7, C to E), significantly more liver TRM cells and TEM were induced than a single vaccination (Fig. 7, C to D), and all boosted mice were protected from challenge with 200 sporozoites on day 66 and then 3000 sporozoites on day 85 (Fig. 7E). Substantial protection at day 85 was also seen in the singly primed groups, suggesting that the endogenous liver TRM cell response was potent and long-lived. To further explore the longevity of this response, mice were primed with VOx.FFRK.NVFLP, and liver TRM cell numbers were tracked for 200 days (Fig. 7F). This showed almost no decay in liver TRM numbers out to this late time point, indicating a half-life of ~425 days (95% confidence interval 195-infinity). Consistent with the maintenance of high TRM cell numbers, 90% of mice remained protected from sporozoite challenge at this late time point (Fig. 7G).

Fig. 7 Conjugate vaccines expand endogenous T cells that provide long-term protection.

(A) B6 mice were vaccinated intravenously with VOx.FFRK.NVFLP, and organs were harvested on day 35 and assessed for NVF-specific CD8+ T cells using tetramers (left). Memory cell phenotype of tetramer+ cells (right). (B) Similar groups were challenged with 200 sporozoites on day 42, and parasitemia was assessed. Surviving mice were rechallenged with 3000 P. berghei sporozoites 1 month later, and parasitemia was assessed. (C and D) B6 mice were vaccinated intravenously with VOx.FFRK.NVFLP on day 0 (NVF/−), day 30 (−/NVF), or both days (NVF/NVF), and memory T cell numbers in the liver (C) and spleen (D) were assessed on day 60. Similar mice were challenged with 200 sporozoites on day 66, and protected mice were rechallenged with 3000 sporozoites on day 85. (E) Percentage of mice protected from 200 sporozoites (white) or not (black) or protected from 200 to 3000 sporozoites (gray bars). Absolute numbers are shown above bars. (F) B6 mice were vaccinated intravenously with VOx.FFRK.NVFLP, and livers were harvested at various time points to enumerate NVF-specific TRM cells. (G) Similar mice were challenged with 200 sporozoites at various time points. Percentage of mice protected (white) or not (black). Absolute numbers are shown above bars. (H to J) B6 mice were vaccinated intravenously with VOx.FFRK.NVFLP or 50,000 RAS and challenged 1 month later with 200 sporozoites. Livers were harvested at the time of euthanasia (day 7 after challenge, red circles; day 12, open circles) and assessed for NVF-specific TRM cells (H) or total liver TRM cells (I). Protection data are outlined in (J). Results in (A) to (E) and (H) to (J) are from two independent experiments using four to six mice per group. Results in (F) are pooled from five independent experiments, where each time point is from two to four experiments with three to six mice per experiment for a total of 8 to 19 mice per time point. Results in (G) are pooled from two independent experiments, where each time point is from one to two experiments with five to six mice per experiment. Data shown are means ± SEM or individual mice. TRM cell numbers in (C) were log-transformed and compared by one-way ANOVA with Tukey’s multiple comparisons. TRM cell numbers in (H) and (I) were log-transformed and compared by t tests. Data in (F) were log-transformed, and the slope was analyzed by linear regression analysis. Groups in (J) were compared using Fisher’s exact test. *P < 0.05, **P < 0.01, and ****P < 0.0001.

Last, to compare the conjugate vaccine to the current gold standard malaria vaccine RAS, mice received a single dose of the conjugate vaccine or 50,000 RAS and were challenged with 200 PbA sporozoites 1 month later. Mice that succumbed to sporozoite challenge were euthanized, and their livers were assessed for the number of NVF-specific TRM cells (Fig. 7H, red circles) and the number of TRM cells of any specificity (Fig. 7I, red circles). Mice that did not become parasitemic were considered protected, and their livers were similarly analyzed at day 12 after challenge (Fig. 7, H and I, open circles). NVF-specific TRM cells were 30-fold higher in mice vaccinated with the conjugate vaccine compared with RAS (Fig. 7H), and total liver TRM cells were threefold higher in the conjugate vaccine group (Fig. 7I). Despite having only a single malaria epitope capable of generating liver TRM cells, mice vaccinated with the NVFLP conjugate vaccine displayed superior protection to mice vaccinated with RAS (Fig. 7J).


Here, we show that our glycolipid-peptide conjugate vaccines can generate large numbers of CD8+ liver TRM cells that, in turn, protect mice against liver-stage P. berghei infection. Although a single dose of our most potent vaccine protected most mice from subsequent sporozoite challenge, this protection could be improved by homologous boosting. The capacity to boost suggests limited induction of NKT cell anergy or neutralizing antibodies, which can affect the efficacy of vaccines (3134). Limited NKT cell anergy may relate to the requirement for conjugate processing, favoring presentation by DCs over other cell types such as B cells, known to cause anergy (3436).

One important factor contributing to the success of a vaccine is the longevity of associated immunity. Here, we show that TRM cell responses to a single dose of our optimized vaccine had an impressive half-life of ~14 months, with protection exceeding 200 days. In other studies, liver TRM cells generated by the adoptive transfer of activated T cells or by vaccination with RAS showed much shorter half-lives of ~1 month (11), raising the intriguing possibility that different vaccination regimes may affect TRM cell longevity.

Although formation of liver TRM cells can occur simply as a consequence of T cell activation (11), liver seeding can be enhanced by local antigen presentation (10, 21, 22) or inflammation (10, 23). In the case of our glycolipid conjugate vaccine, it is likely that antigen presentation occurs in the liver and the spleen and that activation of liver-resident NKT cells leads to their release of proinflammatory cytokines triggering bystander hepatitis (37), thus enhancing TRM cell formation. It should be noted that such inflammation is transient and can be minimized by reducing the length of the glycolipid acyl chain while still facilitating liver TRM cell formation.

Looking ahead to translation of our conjugate vaccine, preliminary data are encouraging because a cytomegalovirus (CMV) conjugate vaccine was able to stimulate NKT cell responses by human peripheral blood mononuclear cells, leading to the expansion of CMV-specific CD8+ T cells (19). This vaccine also activated human DCs in an NKT cell–dependent manner and induced CD8+ T cells with a cytotoxic phenotype (38). The relatively inexpensive manufacturing cost and the ability to simply alter the peptide within the vaccine to target various infectious diseases or cancer highlight the promising nature of this approach. Furthermore, this type of vaccine is relatively stable and can be shipped as powders at ambient temperatures.

One potential caveat of this approach is that the proportion of NKT cells in human livers is lower than in mice, raising the question of efficacy in humans. Humans clearly have NKT cells, and there are numerous studies, particularly in the cancer vaccine area, that show some level of utility by harnessing NKT cells for boosting immunity in humans [reviewed in (39)]. α-GalCer has also been administered with influenza vaccines to swine of mixed genetic background (with similar NKT cell frequencies to humans), with improved vaccine efficacy (40). Injection of free α-GalCer was well tolerated in humans in a phase 1 study at all doses tested (41), and our vaccine was effective in mice at doses about two orders of magnitude lower than the highest dose used in that trial. An analog of α-GalCer that stimulates NKT cells with greater potency than α-GalCer was associated with some liver toxicity in humans (42), indicating that NKT cell function can be significant in the human liver. Given that our vaccine is no more toxic than normal α-GalCer in mice and that this toxicity can be tailored by altering the fatty acid chain length, this vaccination approach should be safe yet effective in humans.

In conclusion, we provide evidence that vaccination with a glycolipid-peptide conjugate designed to induce contemporaneous stimulation of NKT cells and peptide-specific CD8+ T cells, results in a favorable environment for the generation of a stable population of liver TRM cells that provide protection from liver pathogens.

Study design

The aim of this study was to examine the efficacy of a glycolipid-peptide conjugate vaccine to generate memory T cell responses, particularly liver TRM cells, a population critical for protection from malaria. In all experiments, mice were randomly assigned to different groups (n = 10 per group) and vaccinated. One month later, typically four mice per group were examined by flow cytometry for the generation of memory T cells, whereas the remaining six mice were challenged with malaria parasites. Each experiment was performed at least twice, unless stated otherwise. The investigators were not blinded when conducting or analyzing the experiments, and no data were excluded. Raw data can be found in table S1.


Contact for reagent and resource sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by W.R.H. (wrheath{at} and G.F.P. (gavin.painter{at}


C57BL/6 (B6), OT-I (43), PbT-I (44), CXCR6-GFP (24), mT/mG (45), CD40−/− (46), and CD1d−/− mice (47) were bred and maintained at the Department of Microbiology and Immunology, University of Melbourne or the Biomedical Research Unit, Malaghan Institute of Medical Research, New Zealand. All mice used were 6 to 12 weeks of age, and littermates of the same sex were randomly assigned to experimental groups. Animals used for the generation of the sporozoites were 4- to 5-week-old male Swiss Webster mice purchased from the Monash Animal Services (Melbourne, Victoria, Australia) and housed at the School of Biosciences, University of Melbourne, Australia.

Ethics statement

All animal experiments were in accordance with the Prevention of Cruelty to Animals Act 1986, the Prevention of Cruelty to Animals Regulations 2008, the National Health and Medical Research Council (2013) Australian code for the care and use of animals for scientific purposes, or the Animal Welfare Act of New Zealand (1999). The protocols were approved by the Melbourne Health Research Animal Ethics Committee, University of Melbourne (ethics protocols IDs: 1513505, 1513470, and 1714302) or by the Victoria University of Wellington Animal Ethics Committee (ethics protocol ID: 23803).

PbA sporozoite infection

PbA parasite maintenance, sporozoite infection, and parasitemia analysis were performed as previously described (11). Mice were considered protected if parasitemia was absent on day 12. For 3000 sporozoite challenges, naïve groups are not shown but were similar in number to the naïve 200 sporozoite challenge groups, and all mice became parasitemic, indicating infection.

Synthesis of peptides

Peptides 5-azidopentanoyl-AAAHSLSNVYDFNLLLERD and 5-azidopentanoyl-FFRKNVYDFNLL were obtained from commercial manufacturer Peptides and Elephants (Hennigsdorf, Germany). The synthesis of 5-azidopentanoyl-FFRKAAAHSLSNVYDFNLLLERD is described in the Supplementary Materials.

Synthesis of glycolipid-peptide conjugates

Full details are provided in the Supplementary Materials.

Solubilization of compounds for biological studies

Solubilization of α-GalCer and α-GalCer–peptide conjugates was achieved by lyophilizing the samples in the presence of aqueous sucrose, l-histidine, and Tween 20 as previously described for the solubilization of α-GalCer (41). Typically, all compounds were reconstituted in water then further diluted in phosphate-buffered saline (PBS) for intravenous administration.

CD8+ T cell transfer and vaccination

Naïve PbT-I CD8+ T cells were isolated by negative selection from the lymph nodes and/or spleen as previously described (10). Enriched naïve CD8+ T cells were counted, and their purity was analyzed by staining with anti-CD8α and anti-Vα8.3 TCR antibodies. Cell counts were adjusted to 2.5 × 105/ml in PBS, and mice were injected intravenously with 200 μl of cell suspension. OT-I cells were isolated from pooled lymph nodes. A portion of OT-I cells were stained with anti-CD8α, Vα2, CD44, and CD62L antibodies to determine the proportion of naïve (CD8+CD44loCD62Lhi) CD8+ T cells. Forty to fifty thousand naïve CD8+ OT-I T cells were then injected intravenously into each mouse across all treatment groups. Mice were injected with naïve OT-I or PbT-I cells intravenously 1 day before vaccination with 600 or 1 × 106 PFU of recombinant PR8-OVA (H1N1) influenza virus, 0.135 nmol of α-GalCer, or 0.135 nmol of conjugate vaccines intravenously. Naïve PbT-I cells were primed in B6 mice by intravenous injection of 5 nmol of CpG 2006-21798 (10, 48) and 8 μg of an anti-Clec9a antibody genetically fused to LSNYVDFNLLLERD (10, 31), termed anti–Clec9a-NVY. In some experiments, mice receiving anti–Clec9a-NVY were additionally treated with 2.5 × 109 copies of AAV-NVY (10). Mice were vaccinated with RAS as previously described (11).

Lymphocyte isolation from organs

Liver and splenic lymphocytes were isolated as previously described (11).

Flow cytometry

Lymphocytes were stained with NVF-specific tetramers for 1 hour at room temperature before staining with monoclonal antibodies for CD49a (Ha31/8) and NK1.1 (PK136) from BD (North Ryde, NSW, Australia); CD8α (53-6.7), KLRG1 (2F1), Ly5.1 (A20), Ly5.2 (104), CD8α (53-6.7), CD44 (IM7), Ly6C (HK1.4), TCRβ (H57-597), CXCR6 (SA051D1), CXCR3 (CXCR3-173), CX3CR1, and (SA011F11) from BioLegend (Australian Biosearch, Karrinyup, WA. Australia); and CD62L (MEL-14), CD101 (Moushi101), and CD69 (H1.2F3) from eBioscience (Jomar Life Research, Scoresby, VIC, Australia). Dead cells were excluded by propidium iodide staining or far red LIVE/DEAD fixable dye (Thermo Fisher Scientific). In some experiments, cells were treated with Fc block (2.4G2) for 10 min before staining with an α-GalCer–loaded (PBS-44, a gift from P. Savage, Brigham-Young University, UT, USA) CD1d tetramer produced in-house (49) at 4°C for 30 min. Cells were washed before further surface antibody staining for 10 min at 4°C. Antibodies used in combination with CD1d tetramer staining were CD3 (17A2), CD45R/B220 (RA3-6B2), NK1.1 (PK136), and CD69 (H1.2F3) (all from BioLegend, CA, USA), and the viability dye used was 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, NZ). Single color–positive control samples were used to adjust compensation, and cells were analyzed by flow cytometry on a LSRFortessa (BD Biosciences) or LSRII SORP using FlowJo software (Tree Star Inc.).

Intravital 2 photon microscopy

Three days before imaging, recipient CXCR6-GFPKI/+ mice were treated intravenously with 100 μg of anti-CD8 antibodies (clone 2.43). Intravital microscopy was performed on an FVMPE-RS multiphoton microscope (Olympus) as previously described (11). For the generation of movies, image sequences exported from Imaris were composed in Adobe After Effects.

Serum ALT measurements

Measurement of serum ALT levels was performed with a modular analyzer (Roche/Hitachi Modular P800, Roche Diagnostics, IN) by Gribbles Veterinary Clinic (Hamilton, New Zealand) according to a standard operating procedure approved by International Accreditation New Zealand.

Quantification and statistical analysis

Figures were generated using GraphPad Prism 7. Data are shown as mean values ± SEM as indicated in the figure legends. Data were log-transformed and assessed for normality, and then a one-way analysis of variance (ANOVA) with Sidek’s or Tukey’s multiple comparison test or Student’s t test was performed. To compare survival after challenge, groups were compared using Fisher’s exact test. The statistical tests performed on the data are indicated in the figure legends and results, along with sample size indicating the number of animals used. *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001 was considered statistically significant.


Fig. S1. Chemical structures of conjugate vaccines.

Fig. S2. FACS gating strategy to detect memory CD8+ T cell populations.

Fig. S3. Glycolipid-peptide vaccination induces greater numbers of liver TRM cells than a relatively high dose of PR8-OVA.

Fig. S4. Conjugate vaccines lacking the FFRK cleavage sequence do not alter serum ALT levels.

Fig. S5. Processing of vaccines by cathepsin B.

Fig. S6. Generation of memory T cell responses to VOx.FFRK.NVFLP are partially dependent on CD40/CD40L interactions.

Fig. S7. Synthesis of Fmoc-VC-PAB-pNP.

Fig. S8. Synthesis of MaGC-PAB-CV-Fmoc.

Fig. S9. Synthesis of MaGC-PAB-CV-NH2.

Fig. S10. Synthesis of MaGC-PAB-CV-Non.

Fig. S11. Synthesis of V.FFRK.OVALP conjugates.

Fig. S12. Synthesis of SPAAC conjugate vaccine V.FFRK.NVYSP.

Fig. S13. Synthesis of SPAAC conjugate vaccine V.NVYLP.

Fig. S14. Synthesis of SPAAC conjugate vaccine V.FFRK.NVYLP.

Fig. S15. Synthesis of oxime conjugate vaccine VOx.FFRK.NVYSP.

Fig. S16. Synthesis of oxime conjugate vaccine VOx.FFRK.NVFLP.

Fig. S17. Nuclear magnetic resonance (NMR) Spectra of Fmoc-VC-PAB-pNP.

Fig. S18. NMR spectra of MaGC-PAB-CV-Fmoc.

Fig. S19. NMR spectra of MaGC-PAB-CV-NH2.

Fig. S20. NMR spectra of MaGC-PAB-CV-Non.

Fig. S21. High-performance liquid chromatography (HPLC) analysis of V.FFRK.OVALP C-24 vaccine.

Fig. S22. HPLC analysis of V.FFRK.OVALP C22 vaccine.

Fig. S23. HPLC analysis of V.FFRK.OVALP C20 vaccine.

Fig. S24. HPLC analysis of V.FFRK.OVALP C18 vaccine.

Fig. S25. HPLC analysis of V.FFRK.OVALP C8 vaccine.

Fig. S26. HPLC analysis of V.FFRK.OVALP C4 vaccine.

Fig. S27. HPLC analysis of V.FFRK.NVYSP vaccine.

Fig. S28. HPLC analysis of V.NVYLP vaccine.

Fig. S29. HPLC analysis of V.FFRK.NVYLP vaccine.

Fig. S30. HPLC analysis of VOx.FFRK.NVYSP vaccine.

Fig. S31. HPLC analysis of VOx.FFRK.NVFLP vaccine.

Table S1. Raw data.

Movie S1. Imaging of NKT cells within the liver of naïve and vaccinated mice.

Movie S2. Imaging of PbT-I TRM cells within the liver of mice vaccinated with V.FFRK.NVYSP.


Acknowledgments: We thank the BRF facility at the Peter Doherty Institute and the BRU at the Malaghan Institute of Medical Research for their technical support. Funding: This work was supported by the Australian Research Council (CE140100011), National Health and Medical Research Council (NHMRC; 1113293), New Zealand Ministry of Business Innovation and Employment (RTVU1603), and Avalia Immunotherapies. K.K., D.I.G, and W.R.H. are NHMRC Senior Principal Research Fellows (1117766 and 1154457). Author contributions: L.E.H. performed the experiments, analyzed the data, and wrote the manuscript. Y.C.C., M.N.M., T.L.O., S.T.S.C., J.M., J.L., K.J.F., R.S., and C.F.A. performed the experiments and analyzed the data. J.L., L.K., Z.W., L.B., M.H.E., S.G., R.M., T.M.S., D.F.-R., and A.-M.V.-H. performed the experiments. R.J.A, S.L.D., and B.J.C. synthesized, purified, and characterized the vaccines. K.T., P.B., D.G.B., A.C., V.M., G.I.M., I.C., and M.H.L., provided reagents. S.J.T., K.K., and D.I.G. conceived the experiments. I.F.H., G.F.P., and W.R.H. conceived the experiments and wrote the manuscript. All authors contributed to reviewing and editing the manuscript. Competing interests: The authors declare the following competing financial interest(s): G.F.P. and I.F.H. are the chief technical officer and chief scientific officer, respectively, of biotech start-up Avalia Immunotherapies Limited, and D.I.G. and W.R.H. are members of its Scientific Advisory Board. Availa holds exclusive, worldwide license to patents related to aspects of the chemical design reported here. Avalia partially funded the study. R.J.A., B.J.C., D.I.G., S. A. Gulab, W.R.H., I.F.H., L.E.H., and G.F.P. are inventors on patent application (U.S. patent no. 62/846,327) submitted by Victoria University of Wellington, Malcorp Biodiscoveries Limited, and Victoria Link Limited (D.I.G., W.R.H., and L.E.H.) that covers the production of tissue-resident memory T cells with glycolipid-peptide vaccines. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. PbT-I transgenic mice are available from W.R.H. under a material transfer agreement (MTA) with the University of Melbourne. Anti-mouse Clec9A antibodies are available from M.H.L. under an MTA with Monash University. Individual MTAs will be prepared based on the nature of the proposed research.

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