Fig 2: CD19-targeted polymersomes target DLBCL cells in vivo. OCI-Ly8 DLBCL cells were engrafted in NSG mice on day 0 either intravenously (A,B) or subcutaneously (C,D). On day 6, the mice were treated intravenously with either PBS (gray) or αCD19-PSOMcalcein (green). On day 7, the mice were sacrificed, and single-cell suspensions of bone marrow or a solid tumor were analyzed by flow cytometry. Representative histograms are shown with axis-zoomed inlays where appropriate. (A,B) In the bone marrow of mice with intravenously engrafted DLBCL, (A) αCD19-PSOMcalcein was detectable in DLBCL cells (CD19+CD20+) but (B) not in other cells of the bone marrow. (C,D) In the tumors of mice with subcutaneously engrafted DLBCL, (C) αCD19-PSOMcalcein could be detected in DLBCL cells (CD19+CD20+). (D) Most CD19– cells were unaffected by treatment with αCD19-PSOMcalcein, except for a small subset of cells, likely macrophages.

Fig 3: The majority of HELIOS+ CD8 T cells are antigen-experienced. Frozen PBMC were thawed and rested for 2h at 37°C with DNase. Additionally, CD38 was labeled for healthy donors and COVID-19 patients. Ovarian cancer patient samples were also labeled with CD19, CD20, CD33, and CD326. Following overnight fixation and permeabilization, intracellular staining for HELIOS was performed at 4°C. Ki-67 staining was also performed for healthy donors and COVID-19 patients. The samples were analyzed by flow cytometry. (A) Representative donor’s data showing the prevalence of HELIOS+ cells within living CD2+ CD8β+ cells (B) data for 58 donors. These data were obtained from 13 females and 45 males. The raw data are also available in Supplementary Table 1 . The mean frequencies of HELIOS+ cells were 26% and 20% respectively. These two mean frequencies are not different according to an unpaired t-test. (C) Distribution of living CD2+ CD8β+ HELIOS+ cells among T cell subsets is shown for one representative donor and (D) for 51 donors. (E) The prevalence of HELIOS+ cells in living CD2+ CD8β+ cells is compared between healthy donors and COVID-19 patients and (F) between blood and tumors from ovarian cancer patients. The average age of cancer patients was 61 years old, while the average was 55 for the blood donors. These averages are not different according to an unpaired t-test. (G) Frequency of Ki-67+ cells and (H) CD38+ cells among living CD2+ CD8β+ cells are shown in healthy donors and COVID-19 patients. (I) Percentage of Ki-67-positive cells in HELIOS+ and HELIOS- CD8 TILs from ovarian cancer samples. (J) Representative donors’ data showing the TOX expression in non-naïve CD8 T-cells from blood or tumor, and gating strategy to identify TOXhi cells. (K) Percentage of TOXhi cells in HELIOS- or HELIOS+ non-naive CD8 T-cell from tumor samples. Data from 23 donors. Mean +/- SEM are shown. P values * = < 0,05, ** = < 0,01, *** = < 0,001 (one-way ANOVA).

Fig 4: Immunophenotyping of SLE vs. HD BM. BM derived from 9 SLE (○) and 7 HD (⚫) analyzed by flow cytometry. (A) Putative differentiation cascade of ASC. A, B and D represent populations from gating in Figure 1B . Figure made with BioRender.com (B) Flow cytometry gates A (purple), B (blue), C (orange), and D (red) representing BM PC populations for HD and SLE backgated onto CD19 vs CD138 plot. (C) Frequency of BM populations A, B, C, and D as a % of live single cells. (D) Immunophenotyping of immunoglobulin isotypes (IgG, IgA, IgM) and markers (CD28, CD20, and Ki-67) as % of BM fraction A, B, C or D. Error bars indicate mean ± SEM. Statistics calculated as non-parametric Mann-Whitney Test between SLE and HD groups with q-values reported utilizing a two-stage set up Benjamini, Krieger, and Yekutieli correction.

Fig 5: Induction of Robust T Cell Activation in GCs with HIV-1 Env Formulated in CAF01 and MPLA +QS-21 Adjuvants.(A) Overview of experimental design for in-situ proteomics and transcriptional analysis. (B) Representative images of GC from FFPE sections of inguinal LN at baseline and week 2 post protein 1 immunization with HIV-Env gp140 | MPLA +QS-21; scale bar; top, 3 mm, close up, 300 μm. LN sections were stained for CD20 (blue), CD3 (yellow), Ki-67 (magenta), and DNA (gray) to identify GCs. Circular ROIs (100 μm in diameter, total 60) were selected based on co-localization of CD3 with CD20 +Ki-67+GC B cells for proteomic profiling with a 32-plex antibody cocktail. Normalized protein expression (NPE) values were calculated using three negative control IgG probes. (C) NPE of key lineage markers across all ROIs at BL, P1w2, and P2w3. (D–E) Volcano plots show proteins induced post boost in each vaccine group. (F) Violin plots show common proteins induced post protein boost in MPLA and CAF01 groups. (G) Violin plots of proteins induced with CAF01 and MPLA. (H) Volcano plot (left) and Violin plots (right) of proteins significantly different across HIV-1 Env gp140 MPLA +QS-21 and CAF01 regimens. Differential expression was modeled using a linear mixed-effect model to account for the sampling of multiple ROI/AOI segments per patient/tissue. Criterion of significance was nominal p-value <0.05, plots show False discovery rate (FDR).