Fig 2: Quantification of inflammatory cell populations in human liver samples.Immunohistochemical staining was used to detect and quantify changes in inflammatory cell populations between normal and steatotic human liver samples. Positive immunoreactive staining (dark brown) was calculated as a percentage of total area for (A) CD45 positive inflammatory cells, (B) CD68 positive macrophages (C) CD3 positive T lymphocytes (D) CD8 positive cytotoxic T cells and (E) CD56 positive NK cells in frozen sections of normal and steatotic human livers. Results demonstrate a significant increase in the number of CD45 and CD3 positive cells in the steatotic livers when compared to normal liver samples. CD68 macrophages, CD8 cytotoxic T cells, and CD56 NK cells were not significantly altered between samples. Images are 20x.

Fig 3: Steatosis results in changes in the inflammatory cell populations in the murine liver.CD45 positive cells were isolated from normal and steatotic digested liver samples by immunomagnetic beads and then stained for the immune markers CD3 and CD11b before being subjected to Flow cytometric analysis. (A) Diagrams are representative scatter plot of flow cytometric analysis for overall CD45 positive cells in the livers. (B) CD45 positive cells were then gated for the percentage of CD3 positive T lymphocytes or CD11b positive myeloid cells. CD3+ and CD11b+ subpopulations were both increased in the steatotic livers versus the control livers.

Fig 4: Assessing RG7769 binding to T cells in anti‐PD‐1 experienced patients. (A) Comparison of the frequency of NP40435 patients with prior aPD‐1 experience with aPD‐1 naïve patients. (B) Comparison of RG7769 CD3+ T cell receptor occupancy (RO) pre‐ and post‐infusion in NP40435 patients. (C) Control in vitro experiment assessing the effect of PD‐1 competition on RG7769 binding to CD3+ T cells. Activated peripheral blood mononuclear cells (PBMCs) were incubated with IgG4 isotype, nivolumab or pembrolizumab (100 pg/ml➔100 μg/ml), then pulsed with RG7769 (10 μg/ml), and analyzed for T cell anti‐PGLALA‐PE binding. The pooled MFI for PE is shown (data from two independent experiments). (D) Linear regression of max. RG7769 (MFI) to CD3+ T cells in NP40435 anti‐PD‐1+ patients prior to RG7769 infusion (C1D1_pre) as a function of the last reported prior anti‐PD‐1 administration. R 2 = .8338, p = 0.0015. (E) anti‐PGLALA‐PE staining on CD3+ T cells of representative NP40435 patients. Maximum anti‐PGLALA‐PE assessed post ex vivo blood saturation with RG7769, in recently (up to 14 weeks) or historically experienced (>14 weeks) patients versus naïve patients prior to RG7769 (C1D1_pre). Anti‐PGLALA–PE intensities are shown for ex‐vivo saturation and neg. control. (F) CD3+ T cell RG7769 mean ABC in recently (<14 weeks) or historically (>14 weeks) aPD‐1+ patients versus naïve. Data are pooled from (E). ABCs were compared to aPD‐1 naïve (Welch ANOVA test). (G) Schematic of RG7769 binding to T cells in aPD‐1+ patients. Left: RG7769 engages T cells via high‐affinity anti‐PD‐1 engagement if PD‐1 is freely available. Right: In situations where prior aPD‐1 is engaging PD‐1, RG7769 has to displace prior bound aPD‐1 antibodies. Steric hindrance may occur if competing mAbs target an epitope in the PD‐L1 interaction side of PD‐1 [Color figure can be viewed at wileyonlinelibrary.com]

Fig 5: TREG and TFH cells as important factors for vaccine-induced immune responses.Cryopreserved PBMCs isolated from vaccine triple responders and non-responders were stained for surface antigens and intranuclear expression of FOXP3 ex vivo and analyzed by flow cytometry. A Frequencies of TREG cells (CD4+CD127lowCD25+FOXP3+) assessed by flow cytometry (1st study, n = 5 and n = 7 non-responders and responders, respectively, biologically independent samples, single missing values, see Source Data file, day 0 triple non-responders vs. responders p = 0.0390, day 6/7 triple non-responders vs. responders p = 0.0030). Plots show representative tSNE analyses of triple non-responders and responders with the expression density of FOXP3 highlighted (blue = low expression, red = high expression). B Frequencies of TREG cells assessed by TSDR analysis and correlation with TREG cells assessed by flow cytometry before, 3 and 70 days post-vaccination (1st study, n = 5 and n = 7 non-responders and responders, respectively, biologically independent samples, Spearman correlation). C Plot (left) depicting the tSNE analysis of 4 triple non-responders and 4 triple responders (gray) with manually gated TREG cells highlighted and gated accordingly (CD3+CD4+FOXP3+ = purple). tSNE plots of non-responders and responders depicted in pseudo colors indicating low (blue) and high (red) cell densities 3 days post-vaccination. D Mean values of TREG and TFH cell frequencies at the indicated time points post-vaccination normalized to day 0 (1st and 2nd study, n = 10 non responders and n = 15 responders, biologically independent samples). Violin plots show the mean and the quartiles as dashed and dotted lines, respectively. The shape indicated the data distribution. Asterisks denote significant nominal p values as calculated by two-way ANOVA or Mixed-effects analysis for data sets with missing single values (uncorrected Fisher’s LSD, see Source Data file) comparing triple vaccine responders and non-responders at a given time point Source data are provided as a Source Data file.