Fig 2: Loaded NPs enable the robust activation and expansion of endogenous TILs in cancer organoids derived from surgical resections of renal cell, lung cancer, and melanoma tumors. (A) Quantification of live CD3+, CD4+, and CD8+ TILs per 106 organoid cells by FACS analysis after 7-d IgG, anti–PD-1, or NP treatment. (B) qRT-PCR quantification of messenger RNA (mRNA) of IFNG, PRF, and GZMB of FACS-sorted CD3+ TILs from patient-derived organoids (PDOs) after 7-d IgG, anti–PD-1, or NP treatment. (C) The expression of CD69 and CD25 of CD8+ TILs in 7-d cultured melanoma organoids and their production of TNF-α and IFN-γ after ex vivo stimulation with the cell stimulation mixture for 3 h. The flow cytometry plots shown are gated on live CD3+CD8+ TILs. (D) Luminex analysis of the cytokine and chemokine expression from the melanoma organoid culture medium (n = 2 technical replicates for Luminex analysis). The data are represented as log2 fold change of the cytokine concentration over IgG-treated organoids.

Fig 3: Loaded NPs promote self-specific CD8+ T cells expansion and activation in vitro and reduce systemic toxicities of a soluble stimulant mixture in vivo. (A) The TLR1/2 agonist and NOD2 agonist stimulated self-antigen–specific CD8+ T cells in healthy human PBMCs (n = 5). In this assay, PBMCs were directly cultured with Pam3CSK4 and L18-MDP in the presence of pooled self-specific peptides, anti-CD28 Ab, and IL-2 and stained with pooled HLA-A*0201 tetramers at day 7. The T cell activation was characterized by measuring the percentage increase in CD25 expression of self-tetramer+CD8+ T cells. Data are means ± SD. Data were analyzed by unpaired t test with Welch's correction. **P < 0.01. (B) The representative flow cytometric analysis showing the proliferation and activation of self-specific CD8+ T cells from healthy blood donors after stimulation with a mixture of soluble stimulants (Top) or NPs (Bottom) in the presence of pooled self-peptides at day 7.5. The soluble stimulant mixture includes IL-2, Pam3CSK4, L18-MDP, and anti-CD28 Ab. The NPs were loaded with equivalent dose of IL-2, Pam3CSK4, and L18-MDP, and anti-CD28 Abs conjugated to their surfaces. For the proliferation assay, self-specific CD8+ T cells were sorted by pooled HLA-A*0201 tetramers loaded with self-specific peptides, CFSE labeled, and cultured under the different stimulation conditions in the presence of autologous feeder PBMCs with the same peptides used for tetramer sorting. T cell proliferation was characterized by measuring the CFSE dilution of tetramer+CD8+ T cells. The most brightly CFSE-stained cells are not proliferating. For the activation assay, PBMCs were directly cultured under the stimulation conditions in the presence of pooled self-specific peptides and stained with pooled HLA-A*0201 tetramers at day 7.5. The T cell activation was characterized by measuring the percentage increase in CD25 expression of self-tetramer+CD8+ T cells. Data are representative of three human individuals. (C) Representative flow cytometric analysis showing the proliferation of TRP2-specific CD8+ T cells isolated from B16F10 melanoma-bearing mice. CD8+ T cells were sorted from tumor-draining lymph nodes of B16F10 melanoma-bearing mice, labeled with cell trace far red (CTFR), and stimulated with different stimulants such as anti-CD28 Ab plus TRP2 peptide, anti-CD3/CD28 Ab, or NPs plus TRP2 peptide. Data are representative of two independent experiments. (D) The concentration of IL-6 in the serum of C57BL/6 mice 2 h after a single subcutaneous injection of a soluble stimulant mixture or NPs (n = 3). The data were represented as fold change of mean fluorescence intensity (MFI) over the PBS group. (E) Splenomegaly assessment of C57BL/6 mice on day 7 received three injections (day 1, day 3, and day 5) of PBS, NP, or a soluble stimulant mixture. Soluble is short for a soluble stimulant mixture (n = 3). Data are means ± SEM. Data were analyzed by one-way ANOVA with Bonferroni posttest. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 4: Characterization of m-reMAIT cells.(A) Flow cytometric profiles of m-reMAIT cells. mMR1-tet staining and expression of T-cell receptor ß (TCRß), CD4, CD8, CD25, and CD44 and the transcription factors PLZF and ROR?t in m-reMAIT cells on differentiation day 18. (B) 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) dose-dependent activation of m-reMAIT cells. The percentages of CD69+ cells among m-reMAIT cells challenged with the indicated concentration of 5-OP-RU in the presence of WT3 (?), WT3/mMR1 (?), CH27 (?), and CH27/mMR1(¦). (C) MR1-dependent activation of m-reMAIT cells. The percentage of CD69+ cells among m-reMAIT cells cultured with CH27/mMR1, challenged as in (B) in the presence of the anti-MR1 antibody (¦) or the isotype control antibody (?). (D) 5-OP-RU dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with various concentrations of 5-OP-RU. Representative data from two independent experiments are shown. (E) mMR1-tet dose-dependent activation. The percentage of m-reMAIT cells expressing CD69 upon a challenge with the indicated amounts of mMR1-tet. Representative data from two independent experiments are shown. (F) 5-OP-RU- and mMR1-tet-induced cytokines and chemokines. m-reMAIT cells were stimulated with various concentrations of 5-OP-RU (?) or mMR1-tet (¦) and the resultant cytokines and chemokines were quantified with LegendPlex. The concentrations at which each reagent induced a similar degree of activation (% CD69) are shown as relative concentrations (0.1–100 nM for 5-OP-RU and 0.01–10 µg/ml for mMR1-tet). The number on the X-axis corresponds to that in (D) and (E). (G) Tyrosine phosphorylation elicited with 5-OP-RU. A Western blot analysis with PY99 (anti-phosphotyrosine). Upon a challenge with different concentrations of 5-OP-RU for 30 min, the cell lysate from m-reMAIT cells was separated on SDS-PAGE (5 × 105/lane), and subjected to Western blotting. Lane 1, 0; lane 2, 0.1; lane 3, 1.0; lane 4, 10; lane 5, 100; lane 6, 1000; and lane 7, 10,000 (nM). Phosphorylated proteins are indicated with arrows. (H) Time course of tyrosine phosphorylation. A Western blot analysis with PY99. The cell lysate from m-reMAIT cells challenged with 100 nM of 5-OP-RU for the indicated time was separated on SDS-PAGE (5 × 105/lane) and subjected to Western blotting. Lane 1, 0; lane 2, 15; lane 3, 30; lane 4, 60; lane 5, 150; lane 6, 300 (min) . Arrows indicate phosphorylated proteins. (I) Linker for the activation of T cells (LAT) as a phosphorylated 37-kD protein. A Western blot analysis with PY99, anti-LAT, and anti-ß-actin. The cell lysate prepared as described in (H) for 60 min was subjected to Western blotting. A blot with PY99 (upper panel), anti-LAT (middle panel), and anti-ß-actin (lower panel). Phosphorylated LAT and LAT as well as ß-actin are indicated (arrow). Lane 1, 0; lane 2, 0.1; lane 3, 1.0; lane 4, 10; lane 5, 100; lane 6, 1000; lane 7, 10,000 (nM). (J) Tyrosine phosphorylation induced by mMR1-tet. A Western blot analysis with PY99. The cell lysate from m-reMAIT cells challenged with the indicated amounts of unlabeled mMR1-tet for 60 min was subjected to Western blotting (5 × 105/lane). Lane 1, 0; lane 2, 0.43; lane 3, 1.3; lane 4, 4.3; lane 5, 13 (µg/ml). Arrows indicate phosphorylated proteins. MAIT: mucosal-associated invariant T cell. Figure 1—source data 1.Characterization of m-reMAIT cells.The type of antigen-presenting cell (APC), reMAIT cells, and the concentration of 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) (nM) used for activation assays. Activation is shown as the percentage of CD69+ cells among reMAIT cells (B). The percentage of CD69+ MAIT cells challenged with 5-OP-RU in the presence of isotype control or anti-MR1 antibody (in the presence of CH27m as APC) (C). The percentage of CD69+ MAIT cells challenged with 5-OP-RU or mMR1-tet (D, E). Production of the cytokines and chemokines from m-reMAIT cells challenged with 5-OP-RU or mMR1-tet (F). MAIT: mucosal-associated invariant T cell. Figure 1—source data 2.Original gel electrophoresis panels for Figure 1G–J.

Fig 5: PSA protection from HSE is independent of induced Tregs. a % FoxP3+ CD4 Tregs and b CD69+ CD4 T cells in spleen and CLN of PSA or PBS-treated WT mice at day 6 pi. c CD25 expression within FoxP3+ CD4 Tregs in WT mice at day 6 pi, % and mean fluorescence intensity (MFI) in () shown in right top quadrant. d % CD25 within FoxP3+ Tregs (left plot) and FoxP3− CD4+ T cells (right plot), e CD103 expression within FoxP3+ Tregs in WT mice at day 6 pi; % and MFI in () shown in right top quadrant. f CD103 within FoxP3+ Tregs (left plot) and FoxP3− CD4+ T cells (right plot) in the spleen or CLN of WT mice at day 6 pi. Data from three experiments shown. g PSA-treated Treg depleted and control WT mice were monitored for survival after HSV infection and ACV treatment as in Fig. 1a, ns: not significant determined by log rank Mantel–Cox test (n = 11–12 mice). After administration of three (1 week) or six doses (2 weeks) of PSA, MLN in uninfected WT mice were monitored for h cellularity, i % CD4 and CD8 T cells, and j # ICOS+, CD39+, and CD73+ CD4 and CD8 T cells (n = 3 mice); ****p < 0.0001, **p < 0.01 as determined by two-way ANOVA or one-way ANOVA with Sidaks or Turkeys correction, respectively, for multiple comparisons tests. All data show mean ± SEM