Fig 2: pAntp CPP conjugates exhibit enhanced LN accumulation and uptake by APCs in dLNs relative to free peptide antigen. Groups of C57BL/6 mice (n = 3 per group) were immunized subcutaneously with 25 nmol cy5-labeled gp100 or cy5-gp100-pAntp with 25 µg c-di-GMP, and then at selected times inguinal dLNs were isolated for histology or flow cytometry analysis. (A and B) Immunofluorescence of dLNs 48 h after immunization with cy5-gp100 (A) or cy5-gp100-pAntp (B). (Scale bars, 100 µm for main images and 50 µm for Insets.) (C–F) Representative dot plots from day 2 and quantification of percentage of peptide+ cells at 2- or 6-d postvaccination with cy5-gp100 and cy5-gp100-pAntp for (C) CD19+ cells; (D) F4/80+ macrophages; (E) cDC1 cells; and (F) cDC2 cells. **P < 0.01, *P < 0.05, by unpaired Student’s t test. (G) Representative flow cytometry plots showing up-regulation of MHCII and CD86 on CD11c+ DCs in response to the vaccination with the gp100 peptides with or without addition of c-di-GMP adjuvant. (H) Quantification (n = 3 animals per group) of percentage of MHCIIhi/CD86hi DCs in each condition. n.s., not significant by two-way ANOVA with a Bonferroni posttest.

Fig 3: TIM-3 blockade promotes colocalization of cDC1 and CD8+ T cells. (A) Representative histograms showing Venus expression by XCR1+ cDC1 in the spleen (left) and tumor (right) of Xcr1-DTR mice. (B) Representative immunofluorescent microscopy (IF) images showing XCR1+ cDC1 detected in the spleen (left) and tumor (right) of Xcr1-DTR mice, using a polyclonal anti-GFP antibody to detect Venus expression. Scale bars: large image, 100 µm; inset, 50 µm. (C) Flow chart showing the procedure for determining the distance between CD8+ T cells and the nearest cDC1. Following immunofluorescent staining for CD8 and GFP/Venus tumors are scanned and the (x, y) coordinates of each cell is acquired using Definiens TissueStudio analysis software. The (x, y) coordinates are then plotted in MATLAB and used to calculate the distance between each CD8+ T cell and the nearest cDC1. Tumors for each treatment group are then merged, and distances are plotted as a histogram. (D) Kernel density estimate of the distribution of the total T cell population with respect to the distance between a CD8+ T cell and its nearest cDC1. IgG2a/PTX treated tumors shown in red, aTIM-3/PTX treated tumors in blue. Representative image showing nucleus (blue), CD8+ T cells (green), and cDC1s (red) is shown to the right. Scale bar, 100 µm. (E) Quantification of CXCL9 expression by MHCII+CD11c+ splenic cDCs in Xcr1-DTR and Xcr1-DTR/Cxcl9-/- mice. (F) Kernel density estimate of the distribution of CD8+ T cells in Xcr1-DTR/Cxcl9-/- mice. (G) Representative IF stained tumor, showing Hoechst (blue), CD8a (green), XCR1 (red), Ki67 (magenta). Arrow indicates a Ki67+CD8+ T cell. Scale bars: 100 µm, large image; 50 µm, inset. (H) Kernel density estimate of the distribution of Ki67- (green) and Ki67+ (blue) CD8+ T cells in IgG2a/PTX (left, n=28) or aTIM-3/PTX (right, n=34) treated tumors. (I) Kernel density estimate of the distribution of the Ki67+CD8+ T cell population within IgG2a/PTX treated tumors (red) vs aTIM-3/PTX treated tumors (blue). Statistical differences between T cell localization distributions determined by non-parametric Kolmogorov-Smirnov test, with the Kolmogorov-Smirnov D statistic shown. cDC, conventional dendritic cell; DTR, diphtheria toxin receptor; GFP, green fluorescent protein; PTX, paclitaxel; TIM-3, T cell immunoglobulin and mucin domain containing-3.

Fig 4: Blocking PGE2 signaling via the EP2 receptor increases AM numbers in aged mice.a–c Aged (18–22 months) C57BL/6 mice were given 7 daily i.p. injections of an EP2 antagonist. Concurrently, BrdU (0.8 mg/mL) was given in the drinking water ad libitum. AMs (i.e., CD45+ CD11c+ SiglecF+) were then collected from the BALF and analyzed by flow cytometry for BrdU incorporation, indicating proliferation, and Annexin V staining, indicating apoptosis. a Total cell count of lavaged AMs. n = 6/group. b Percentage of AMs that are BrdU+. n = 5/group. c Percentage of AMs that are Annexin V+. n = 6/group. d–f Aged mice were given clodronate-loaded liposomes to deplete resident AMs prior to an adoptive transfer of CFSE+ WT or EP2 KO AMs as depicted in (d). e, f AMs were analyzed by flow cytometry 4 weeks following transfer for CFSE dilution, a marker of proliferation n = 6/group. f Representative histogram of CFSE MFI. For panels a–e, statistical significance analyzed by Mann–Whitney, error bars represent SEM and each point represents a biological replicate. Source data are provided as a Source Data file.

Fig 5: mLN SCs modulate subset composition and transcriptional signature of resident DCs. Indicated LNs were transplanted to the popliteal fossa of SPF-housed mice. Eight to sixteen weeks later, single cell suspensions were generated by enzymatic digestion. a–d Single cell suspensions were analyzed using flow cytometry. a Exemplary dotplot of viable Lin- cells from transplanted LN. (b) Exemplary dotplot of subsets from resDCs and migDCs from indicated LNs. c–d Scatterplot shows frequencies of indicated subsets among migDCs (c) and resDCs (d); data pooled from two to three independent experiments (n = 12–16). e resDCs were isolated from indicated LNs. RNA-seqL and subsequent analysis was performed. Colored numbers in scatterplots represent DEGs for the respective pair-wise comparisons of resDCs (|log2(FC)| = 1 and q-value = 0.05). On the x-axis log2FC of gene expression from resDCs (endogenous LNs) is plotted. On the y-axis log2(FC) of gene expression from resDCs of transplanted mLN-SPF vs. pLN-SPF is plotted. f Heatmap of expression of genes persistently down- (pink dot, left) or up-regulated (blue dot, right) in resDCs isolated from transplanted mLN-SPF. Numbers in brackets indicate average log2(RPKM) expression of respective genes across all experimental groups. Data pooled from two to three independent experiments (n = 3–8). migDC migratory dendritic cell (Lin-CD11c+MHCIIhigh), resDC resident dendritic cell (Lin-CD11chighMHCII+), Lin CD3+CD45R+Ly6G+F4/80high, FC fold-change, RPKM reads per kilobase of exon length per million mapped reads, Endo endogenous, Tx transplanted