Reviewer #2 (Public Review): Vaccination with mycobacterial lipid loaded nanoparticle leads to lipid antigen persistence and memory differentiation of antigen-specific T cells

Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Data availability References Peer review Author response Article and author information Abstract Mycobacterium tuberculosis (Mtb) infection elicits both protein and lipid antigen-specific T cell responses. However, the incorporation of lipid antigens into subunit vaccine strategies and formulations has been underexplored, and the characteristics of vaccine-induced Mtb lipid-specific memory T cells have remained elusive. Mycolic acid (MA), a major lipid component of the Mtb cell wall, is presented by human CD1b molecules to unconventional T cell subsets. These MA-specific CD1b-restricted T cells have been detected in the blood and disease sites of Mtb-infected individuals, suggesting that MA is a promising lipid antigen for incorporation into multicomponent subunit vaccines. In this study, we utilized the enhanced stability of bicontinuous nanospheres (BCN) to efficiently encapsulate MA for in vivo delivery to MA-specific T cells, both alone and in combination with an immunodominant Mtb protein antigen (Ag85B). Pulmonary administration of MA-loaded BCN (MA-BCN) elicited MA-specific T cell responses in humanized CD1 transgenic mice. Simultaneous delivery of MA and Ag85B within BCN activated both MA- and Ag85B-specific T cells. Notably, pulmonary vaccination with MA-Ag85B-BCN resulted in the persistence of MA, but not Ag85B, within alveolar macrophages in the lung. Vaccination of MA-BCN through intravenous or subcutaneous route, or with attenuated Mtb likewise reproduced MA persistence. Moreover, MA-specific T cells in MA-BCN-vaccinated mice differentiated into a T follicular helper-like phenotype. Overall, the BCN platform allows for the dual encapsulation and in vivo activation of lipid and protein antigen-specific T cells and leads to persistent lipid depots that could offer long-lasting immune responses. eLife assessment The authors generate a new formulation built upon a previous nanoparticle platform to generate a new system termed bicontinuous nanospheres (BCN), allowing for the dual incorporation of lipid and protein antigens. The authors generate mycolic acid (MA)-loaded BCN and perform a series of characterization studies to demonstrate the superior performance of this new formulation relative to the original one in terms of antigen persistence, a quality needed to sustain responses after vaccination. This work provides important new insights relevant to the TB vaccine field and it suggests that alternative antigens to proteins could be used in TB vaccine formulations. The data are convincing and will be of interest to individuals working on tuberculosis, vaccines and basic immunology. https://doi.org/10.7554/eLife.87431.3.sa0 About eLife assessments Introduction Vaccination is currently the most effective method for the prevention and eradication of infectious diseases. In particular, the development of subunit vaccine formulations that include only immunodominant antigens from pathogens paired with select adjuvants have contributed to these efforts. In contrast to attenuated and inactivated vaccines that contain diverse molecular components of pathogens, subunit vaccines are often preferred due to their simplicity, safety, stability, and scalability of production (Karch and Burkhard, 2016). Yet the development of effective vaccines for several key endemic parasitic and bacterial pathogens has remained elusive. Antigen selection for subunit vaccines has thus far focused on protein molecules, overlooking a vast family of non-protein antigens such as lipids, glycolipids, metabolites, and phosphoantigens, which can be found on and within pathogens and can be recognized by various subsets of unconventional T cells (Legoux et al., 2017). Persistent antigen exposure is known to occur naturally after infection through antigen archiving by follicular dendritic cells (FDCs), which periodically present antigens to cognate B cells and aid in affinity maturation and somatic hypermutation (Heesters et al., 2014; Hirosue and Dubrot, 2015). Antigen persistence can also support the maintenance of T cell response through the periodic priming of circulating T cells (Takamura et al., 2010; Kim et al., 2010; Demento et al., 2012; Zammit et al., 2006; Woodland and Kohlmeier, 2009; Vokali et al., 2020). Subcutaneous vaccination has also been shown to lead to the archiving of protein antigens within lymphatic endothelial cells (LECs), which induce the development of protective T cell immunity (Tamburini et al., 2014; Walsh et al., 2021). However, little is known about how other vaccination routes and vaccine formulations may affect antigen archiving and persistence. Lipid and glycolipid antigens can be presented by group 1 (CD1a, b, and c) and group 2 (CD1d) CD1 molecules to CD1-restricted T cells. Unlike MHC molecules, CD1 molecules exhibit limited polymorphism (Reinink and Van Rhijn, 2016). Therefore, CD1-restricted microbial lipid antigens are likely to be recognized by most individuals, making them attractive targets for vaccine development (Joosten et al., 2019). Group 1 CD1-restricted T cells have diverse T cell receptors and can recognize a variety of lipid and glycolipid antigens (Morgun et al., 2021). Microbial lipid-specific group 1 CD1-restricted T cells have been identified from several pathogens, including Mtb and other mycobacterial species, Staphylococcus aureus (SA), Borrelia burgdorferi, Salmonella typhimurium, and Brucella melitensis (Morgun et al., 2021). CD1b-restricted T cells specific to Mtb lipids including mycolic acid (MA), an immunodominant lipid antigen (Busch et al., 2016) and the component of the mycobacterial cell wall, have been identified in patients with tuberculosis (TB) (Lopez et al., 2020; Montamat-Sicotte et al., 2011). As group 1 CD1 molecules are not expressed in mice, we have previously developed a human CD1 transgenic mouse model (hCD1Tg). (Felio et al., 2009) Using hCD1Tg mice, we have shown that transgenic CD1b-restricted DN1 T cells specific for MA could protect against Mtb infection (Zhao et al., 2015), making these potential antigens of interest in a subunit vaccine against TB. The primary goal of a vaccine is to induce the differentiation of the adaptive immune system such that upon re-exposure to the antigen, a faster and larger response occurs, halting pathogen spread. While conventional T cells and B cells are known to undergo this differentiation, unconventional T cells such as NKT cells do not possess a comparable memory phenotype. The presence of Mtb lipid-specific group 1 CD1-restricted memory T cells has been described in both human and mice (Lopez et al., 2020; Montamat-Sicotte et al., 2011; Felio et al., 2009). However, the properties of memory group 1 CD1-restricted T cells, which share a similar developmental pathway as NKT cells, are not known. Lipid antigens are difficult to formulate and administer due to their hydrophobic nature. To assess the utility of MA as a component of a subunit vaccine, we previously encapsulated MA in poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-b-PPS) micellar nanocarriers (MA-MC) (Shang et al., 2018). Nanocarriers composed of PEG-b-PPS possess several unique benefits compared to other delivery platforms, including high stability in aqueous solution (Bobbala et al., 2020), minimal background immunostimulation (Burke et al., 2022), and triggered release of cargo when exposed to physiological levels of reactive oxygen species (Du et al., 2017; Scott et al., 2012; Napoli et al., 2004). We showed that MA-MC could effectively activate adoptively transferred DN1 T cells and elicit polyclonal group 1 CD1-restricted T cell response in hCD1Tg mice after intranasal delivery (Shang et al., 2018). While effective for stable loading of lipophilic and amphiphilic payloads, micellar nanocarriers, do not allow for facile encapsulation of hydrophilic payloads like protein antigens. Here, we build upon our prior lipid antigen vaccine formulation by employing a nanocarrier platform engineered for the dual loading of both lipid and protein antigens. BCN are polymeric analogs to lipid cubosomes and thus possess a highly organized internal cubic organization containing both intertwined hydrophobic bilayers for lipid loading as well as hydrophilic aqueous channels for protein loading (Allen et al., 2019). Using the flash nanoprecipitation (FNP) technique (Saad and Prud’homme, 2016; Bobbala et al., 2018), we fabricated MA-loaded BCN (MA-BCN) and found them to have the superior stimulatory capacity in vivo compared to MA-loaded poly(D,L-lactide-co-glycolide) (PLGA) nanocarriers (MA-PLGA), which is a widely used nanocarrier platform for Mtb vaccine development (Lin et al., 2018; Khademi et al., 2018). Furthermore, we found that after MA-BCN vaccination, MA could persist for 6 weeks post-vaccination within alveolar macrophages, a phenomenon which was dependent on the presence of an encapsulating vector, nanocarrier, or bacterial, but not the route of vaccination. Due to the enhanced stability of the BCN architecture that can support the loading of diverse and multiple payloads (Bobbala et al., 2020; Modak et al., 2020), we were able to efficiently co-encapsulate within BCN both MA and Ag85B, an immunodominant protein antigen of Mtb (Huygen, 2014). Interestingly, while both antigens activated their corresponding antigen-specific T cells, only MA resulted in antigen persistence, suggesting a potentially unique characteristic of subunit vaccines containing lipid antigens. Results BCN morphology preserved after MA loading We scalably assembled spherical BCN with and without loaded MA using FNP as previously described (Bobbala et al., 2018). Dynamic light scattering (DLS) showed that both MA-BCN and BCN were consistent in size (364±19 nm and 354±14 nm, respectively), and were monodisperse based on their polydispersity indices (PDIs) of 0.24±0.04 and 0.21±0.05, respectively (Figure 1A). We next verified that the BCN formulation maintained its characteristic interconnected aqueous channels using cryogenic transmission electronic microscopy (cryo-TEM) (Figure 1B) and negative staining transmission electron microscopy (TEM) (Figure 1C). Using small angle X-ray scattering (SAXS) studies, we confirmed the internal cubic organization of BCN. Bragg peaks at the √2, √4, and √6 ratios show that the primitive type of cubic internal organization was preserved between MA-BCNs and BCNs (Figure 1D). Thus, MA encapsulation did not disturb the BCN architecture. We also manufactured a PLGA nanocarrier formulation (PLGA-NP) with and without MA encapsulation. PLGA-NP morphology was confirmed using cryo-TEM (Figure 1—figure supplement 1A) and SAXS (Figure 1—figure supplement 1B) and the size of blank PLGA-NP and MA-loaded PLGA-NP (MA-PLGA) were found to be 169±4 and 163±5 nm, respectively, with PDIs of 0.18±0.04 and 0.19±0.12, respectively (Figure 1E). Using the coumarin derivatization method (Shang et al., 2018), we found that BCNs could more efficiently encapsulate MA than PLGA-NPs (Figure 1F). We previously noted that the highly stable cubic architecture of BCN can lead to the retention of cargo within the endolysosomal compartment (Bobbala et al., 2020). Indeed, Texas Red-Dextran loaded BCN were mostly found in lysosomes while Texas Red-Dextran loaded PLGA-NP could be found both within the lysosome and cytosol of cells (Figure 1—figure supplement 2A, B). Figure 1 with 2 supplements see all Download asset Open asset Physicochemical characterization of bicontinuous nanospheres (BCN) and PLGA nanocarrier formulation (PLGA-NP). (A) Dynamic light scattering (DLS) analysis of blank BCN and MA-loaded BCN (MA-BCN). (B) Cryo-TEM of MA-BCN (scale = 500 nm). (C) Negative staining transmission electron microscopy (TEM) images of MA-BCN. (D) Small angle X-ray scattering (SAXS) of blank BCN and MA-BCN. (E) Dynamic light scattering (DLS) analysis of blank poly(D,L-lactide-co-glycolide) (PLGA) and MA loaded poly(D,L-lactide-co-glycolide) (MA PLGA). (F) MA encapsulation efficiency for MA-BCN and MA PLGA. N=3 per condition. Data represented as mean ± SD. MA-BCN activates MA-specific T cells in vitro and in vivo As PLGA has served as a component of a wide range of vaccine formulations, including Mtb vaccines (Lin et al., 2018; Khademi et al., 2018), we benchmarked PEG-b-PPS BCN against PLGA-NP as a nanocarrier platform for lipid antigens. Both MA-BCN and MA-PLGA were highly effective in activating both mouse (DN1) and human (M11) MA-specific T cells in vitro and inducing IFN-γ production (Figure 2A–D). In particular, MA-BCN were significantly better at activating MA-specific T cells compared to free MA at equivalent concentrations (Figure 2A, B and D). In comparison to MA-BCN, MA-PLGA was significantly more effective at stimulating mouse MA-specific T cells in vitro at lower concentrations. Interestingly, even in the absence of MA, PLGA-NP showed strong stimulation, particularly for M11 T cells. These results demonstrate that MA encapsulation within BCN enhances its ability to stimulate antigen-specific T cells and reveal a considerable background, and thus difficult to control, the stimulatory effect of blank PLGA-NP. In contrast, blank BCN showed little background immunostimulation (Burke et al., 2022), allowing a more controlled dose-dependent increase in T cell responses as the loaded MA concentration increased. Figure 2 Download asset Open asset MA-loaded bicontinuous nanospheres (MA-BCN) effectively activated MA-specific T cells in vitro and in vivo. (A, B) Bone marrow-derived dendritic cells (BMDCs) were pulsed for 18 hr. with selected nanoparticles at various concentrations, co-cultured for 48 hr. with DN1 T cells, and T cell activation was measured. (A) Percentage of CD69 and CD25-expressing DN1 T cells. (B) IFN-γ production measured by enzyme-linked immunosorbent assay (ELISA). (N=3). (C, D) ELISPOT of IFN-γ from MA-specific human M11 T cells cultured for 16 hr with monocyte-derived dendritic cells with selected nanoparticles at high (C) and low (D) concentrations. Data are representative of two independent experiments and displayed as mean ± SEM. Statistical analysis: two-way ANOVA, significance designated for MA-BCN vs free MA. (E–G) hCD1Tg mice were IN vaccinated with selected nanoparticles and CellTrace Violet-labeled DN1 T cells were adoptively transferred the next day. After 1 week, DN1 T cell activation and proliferation were measured. (E) Representative FACS plots of DN1 T cells in the lymph nodes (LN). Percentage of proliferating (F) and CD44-expressing (G) DN1 T cells in the LN and lung. Data represented as mean ± SEM. Statistical analysis: two-way ANOVA. *p<0.05, p<0.01, *p<0.001, ****p<0.0001. We next tested the ability of MA-loaded and unloaded BCN and PLGA to stimulate DN1 T cells in vivo. Intranasal (IN) vaccination of hCD1Tg mice was followed by the adoptive transfer of DN1 T cells. Surprisingly, we found that vaccination with MA-BCN induced a significantly higher percentage of proliferation and activation in DN1 T cells than MA-PLGA in the draining lymph nodes (LN) and lungs at 1 week post-vaccination (Figure 2E–G). The extent of cell proliferation induced by MA-PLGA was not significantly different from that of blank PLGA (Figure 2F). Since MA-BCN effectively stimulated MA-specific T cells in vivo while MA-PLGA did not, we focused on further characterizing and assessing vaccination via solely MA-BCN formulations. MA persists while Ag85B does not after vaccination with Ag85B-MA-BCN Given the lack of knowledge regarding BCN and lipid antigen kinetics, it would be of interest to test whether delivered antigen could persist in the mouse weeks post-vaccination (Figure 3A). We switched from intranasal vaccination to intratracheal (IT) vaccination, as it allowed for the reliable delivery of a larger volume of the vaccine. We found that a significant proportion of the adoptively transferred DN1 T cells could proliferate and become activated in the LNs, lung, and spleen of MA-BCN-vaccinated mice 6 weeks post-vaccination (Figure 3B–D). This activation and proliferation were MA-specific, as no activation and proliferation of Ag85B-specific p25 Tg (p25) T cells were observed (Tamura et al., 2004). Figure 3 Download asset Open asset Vaccination with MA-bicontinuous nanospheres (MA-BCN) leads to antigen persistence 6 weeks post-vaccination. hCD1Tg mice were IT vaccinated with MA-BCN or BCN. 6 weeks later, CellTrace-labeled p25 and DN1 T cells were adoptively transferred, and T cell activation was measured after 1 week. (A) Experimental design. (B) Representative FACS plots of p25 and DN1 T cells in the lymph nodes (LN). Percentage of proliferating (C) and CD44-expressing (D) p25 and DN1 T cells in the LN, lung, and spleen. N=3 or 6 per condition. Outliers were identified through the Grubbs method with alpha = 0.05 with one sample removed. Data represented as mean ± SEM. Statistical analysis: two-way ANOVA. *p<0.05, p<0.01, *p<0.001, ****p<0.0001. Since BCN allows for the co-loading of lipid and protein antigens, we assessed the ability of dual-loaded Ag85B-MA-BCN to activate antigen-specific T cells as well as lead to antigen persistence. We co-loaded Ag85B and MA in BCN and found Ag85B-MA-BCN to have a diameter of 380 nm and PDI of 0.223, comparable to MA-BCN (Figure 4A). Encapsulation efficiency was also relatively high at 70%. Vaccination of hCD1Tg mice with Ag85B-MA-BCN activated and induced proliferation of both p25 and DN1 T cells in the LN, lung, and spleen (Figure 4B–D) at 1 week post-vaccination. In addition, DN1 T cells were still able to be activated 6 weeks post-vaccination in the context of an Ag85B-MA-BCN vaccination, while p25 T cells did not show increased activation or proliferation compared to blank BCN control at this time point (Figure 4E–H). Therefore, MA, but not Ag85B, appears to persist in the context of an Ag85B-MA-BCN vaccination. Figure 4 Download asset Open asset Ag85B and Mycolic acid (MA) dual-loaded bicontinuous nanospheres (BCNs) activate antigen-specific T cells without Ag85B persistence. (A) Dynamic light scattering (DLS) analysis of blank BCN and Ag85B-MA-BCN. (B–D) hCD1Tg mice were IT vaccinated with Ag85B-MA-BCN or BCN and CellTrace-labeled p25 and DN1 T cells were adoptively transferred the next day. After 1 week, T cell activation and proliferation were measured. (B) Representative FACS plots of p25 and DN1 T cells in the lymph nodes (LN). Percentage of proliferating (C) and CD44-expressing (D) p25 and DN1 T cells in the LN, lung, and spleen (N=4 or 5). (E–G) hCD1Tg mice were IT vaccinated with blank BCN or Ag85B-MA-BCN. 6 weeks later, CellTrace-labeled p25 and DN1 T cells were adoptively transferred, and T cell activation was measured after 1 week. (E) Representative FACS plots of p25 and DN1 T cells in the LN. (F) Percentage of proliferating p25 and DN1 T cells. (G) Percentage of CD44-expressing p25 and DN1 T cells in the LN and lung. (N=4). Data represented as mean ± SEM. Statistical analysis: two-way ANOVA. ns = not significant, *p<0.05, p<0.01, *p<0.001, ****p<0.0001. MA persistence was dependent on encapsulation but not route of delivery or delivery vector To determine whether BCN encapsulation contributed to MA persistence, we designed an experiment that would allow direct comparison between MA-BCN and free MA. To account for their differential efficiency in stimulating DN1 T cells, we pulsed bone marrow-derived dendritic cells (BMDCs) from hCD1Tg mice with MA or MA-BCN at 10 μg/mL and 5 μg/mL MA concentration, respectively (Figure 5A). hCD1Tg mice were immunized with MA or MA-BCN-pulsed BMDCs at either 1 week or 6 weeks before the adoptive transfer of DN1 T cells, allowing comparison of short-term vs. long-term vaccination conditions. We found that while there were no significant differences in DN1 T cell activation and proliferation between mice vaccinated with free MA and MA-BCN pulsed BMDCs at 1 week post-vaccination, after 6 weeks, DN1 T cell response could only be detected in the MA-BCN vaccination condition (Figure 5B, C). Thus, BCN encapsulation contributed to the persistence of MA. We then tested whether the BCN structure could contribute to antigen persistence by comparing PEG-b-PPS BCNs to PEG-b-PPS MCs using a similar experimental setup (Figure 5—figure supplement 1A). We found that MA-BCN and MA-MC overall had similar ability to activate DN1 T cells in both short-term and long-term vaccination time points (Figure 5—figure supplement 1B, C). Thus, the structure of the encapsulating NP did not play a significant role in the antigen persistence of MA. Figure 5 with 2 supplements see all Download asset Open asset Route of vaccination or vector type does not affect antigen persistence. hCD1Tg BMDCs were pulsed with MA-bicontinuous nanospheres (MA-BCN) or mycolic acid (MA) at concentrations of 5 mg/mL and 10 mg/mL, respectively. hCD1Tg mice were IT vaccinated with MA or MA-BCN pulsed bone marrow-derived dendritic cells (BMDCs) at 6 weeks or 1 week prior to the adoptive transfer of CellTrace-labeled DN1 T cells. T cell activation and proliferation were measured 1 week after adoptive transfer. (A) Experimental design. (B, C) Percentage of proliferating (B) and CD44-expressing (C) DN1 T cells in the lymph nodes (LN), lung, and spleen of vaccinated mice. N=4 per condition. hCD1Tg mice were vaccinated SC (subcutaneously) or IV (intravenously) with blank BCN, MA-BCN, or attenuated Mycobacterium tuberculosis (Mtb) strain and 6 weeks later CellTrace-labeled DN1 T cells were adoptively (D) Representative FACS plots of DN1 T cells in the LN of mice vaccinated with conditions. (E) Percentage of proliferating DN1 T cells and (F) Percentage of CD44-expressing DN1 T cells in the LN and spleen of mice vaccinated with MA-BCN and and attenuated per condition. Data represented as mean ± SEM. Statistical analysis: two-way ANOVA. ns = not significant, *p<0.05, p<0.01, *p<0.001, ****p<0.0001. To the IT route of vaccination a role in the development of antigen persistence, we tested intravenous and subcutaneous vaccination routes with MA-BCN. DN1 T cell activation and proliferation were detected at 6 weeks post-vaccination through both routes of vaccination (Figure suggesting that the antigen persistence is not unique to IT vaccination. To assess whether Mtb lipid antigen persistence could also be observed in mice vaccinated with an attenuated strain of we vaccinated hCD1Tg mice SC with Mtb strain and tested for the persistence of both Ag85B and MA after 6 The Mtb strain in mouse studies than but still long-term against Mtb equivalent to et al., 2004). We confirmed that no bacterial remained at the 6 weeks time point (Figure 5—figure supplement to MA-BCN-vaccinated mice, DN1 T cells were activated and in the draining lymph nodes and spleen of mice vaccinated with the attenuated Mtb strain (Figure In with our with BCN, no proliferation was in p25 T cells (Figure 5—figure supplement These results that the incorporation of MA in a nanocarrier vaccine allows for the of the lipid antigen persistence induced by vaccination with an attenuated Mtb vaccine. MA persists within alveolar macrophages in the To the of antigen persistence, we the of BCN after IT vaccination using BCN loaded with the hydrophobic Since is a lipophilic like MA, into the BCN we could the of BCN without to perform We found that were within the at all time points tested 48 6 and 6 (Figure We were able to through both in vivo system and at 6 weeks post-vaccination that was significantly control (Figure upon into aqueous suggesting that structure remained at this time analysis (Figure supplement that BCN was found within alveolar macrophages at both and time points with and at time points and (Figure Figure 6 with 2 supplements see all Download asset Open asset Persistent mycolic acid (MA) in due to encapsulation alveolar macrophages and activates T cells through or hCD1Tg mice were IT vaccinated with bicontinuous nanospheres (BCN) loaded with a hydrophobic at 6 weeks, 6 48 or 4 hr prior to the nodes (LN), lung, and spleen were using (A) In was then and presence of BCN was in (B) lung, and (C) cells, alveolar macrophages B cells T cells cells by hCD1Tg mice were IT vaccinated with either BCN or MA-BCN. After 6 weeks, alveolar macrophages were from the lungs using or cells were co-cultured with DN1 T cells in the presence or absence of bone marrow-derived dendritic cells (BMDCs) for 48 and DN1 T cells activation was measured. (D) Experimental design. (E) Percentage of CD25-expressing DN1 T cells. Data represented as mean ± SEM. Statistical analysis: two-way ANOVA. ns = not significant, *p<0.05, **p<0.01, We next tested whether MA likewise within 6 weeks after vaccination, we for using a cell system containing which is highly expressed in and a small of in the (Figure We co-cultured or the through with or without BMDCs and DN1 T cells (Figure supplement While both and from MA-BCN vaccinated mice with BMDCs could activate DN1 T cells (Figure with to significantly higher DN1 T cell suggesting are the primary of MA persistence. The ability of through to likewise lead to DN1 T cell activation suggests MA may also persist in other cell or could be to in Furthermore, could not present MA to DN1 T cells, noted by the lack of T cell activation in the absence of suggesting that MA may be transferred from to for In CD1b is not expressed on (Felio et al., 2009; and the presence of may be both in vitro and in vivo for T cell activation to These data that MA-BCN persists within in the after IT vaccination and are for antigen and activation of MA-specific T cells. Vaccination leads to DN1 T cells into T follicular helper-like T cells To the memory of DN1 T cells after MA-BCN vaccination, we a DN1 bone mouse model adoptively transferred DN1 T cells in mice long-term (Figure After vaccination, the of DN1 T cells in the blood increased and remained high the time point of (Figure memory T cell subsets have been 1 after the antigen the DN1 T cell 6 weeks post-vaccination. We found that the most memory within DN1 T cells were used to memory T cells, particularly in the LN (Figure and Figure supplement 1A). To the memory DN1 T cells, we analysis on and DN1 T cells from of MA-BCN vaccinated mice. We found that memory and DN1 T cells after component analysis these from the (Figure supplement of expressed were identified of which and in the memory (Figure supplement 1C). we which T cell memory DN1 T cells most this we data from the differentiated memory memory memory and two and compared the Using we found that memory DN1 T cells most to and (Figure and when this was most to cells (Figure Figure with 1 supplement see all Download asset Open asset MA-bicontinuous nanospheres (MA-BCN) vaccination leads to DN1 T cell