Fig 2: Single cell sorting of gp140 trimer specific memory B cell from PBMC of HIV infected patient. (A) gate on lymphocyte; (B) gate on IgM (PerCP-cy5.5) negative, 7AAD (PerCP-cy5.5) negative, CD3(PE-CY7) negative and CD14 (PE-CY7) negative population; the purpose is to remove IgM positive cells and T cells. (C) Gate on CD19 (FITC) positive and IgG (Pacific blue) positive population; the purpose is to gate the IgG positive B cells. (D) Gate on SF162gp140 trimer binder (PE), the purpose is to fish out antigen specific IgG positive B cells.

Fig 3: Ad-Cab induces 4T1 tumor control in vivo in presence and absence of CD8+ T cells. (A) Schematic diagram of treatment schedule for 4T1 bearing mice. Mice were treated either with PBS (mock), Ad-5/3Δ24, Ad-Cab or mPD-L1 after 7 days postengraftment of 3×105 cells in the right flank of the mice. Treatments were given on days 7, 9, 11, 13,17, 19 and 21. Ad-5/3Δ24 and Ad-Cab were given intratumorally at a dose of 1×109 viral particles while 100 µg of mPD-L1 was given intraperitoneally. (B) Summary data for average tumor growth for all treatment groups for 4T1 tumor model. (C) Granulocytic (CD11b+Ly6 Ghi Ly6C-) and monocytic (CD11b+Ly6 Chi Ly6G-) MDSC infiltration in 4T1 tumor microenvironment. Cell percentages of granulocytic (D), monocytic (E) MDSC and TAM (F). NK cell activation (G, H) and CD8 (I) cell activation was determined using the CD107a degranulation marker. CD8+ T cell exhaustion was also measured using PD1 (J). (K) Schematic diagram of treatment (black arrows) and CD8 depletion (red arrows) schedule for 4T1 bearing mice. Treatment scheduled was the same as previously but 1 day before treatment 500 µg of CD8 depleting antibody was given and then every 2 days 100 µg was given. (L) CD8 and CD4 cell staining on CD3 gated peripheral blood from mice treated with or without depleting CD8 antibody. (M) Summary data for average tumor growth for all treatment groups for CD8 depleted 4T1 tumor model. (N) Kaplan-Meier survival curve for the treatment groups for CD8 depleted 4T1 bearing mice. MDSC, myeloid-derived suppressor cell; TAM, tumor-associated macrophage.

Fig 4: Dynamic regulations of GITR and PD1 expression on TILs from human CRC(A) Experimental design.(B–G) Transcriptomic (B–E) and histological analyses (F–G) on tumor-infiltrating lymphocytes (TILs) from human CRC samples.(B) Uniform manifold approximation and projection (UMAP) representation of CD3+ TIL clusters from 7 CRC patients (top). Fraction of cells in each CD3+ TIL clusters per sample (bottom). p, patient; core, neoplasm core; border, neoplasm border.(C) Heatmap of differentially expressed genes in CD3+ clusters from (B).(D) Diffusion map of CD3+ clusters using the first two diffusion components (top). Clusters are colored according to identities in (B). Pseudotime (bottom left) and feature plots for CD8 and CD4 (bottom right) are depicted on the same scale.(E) Sliding windows (N = 200) of average expression of genes of interest in CD8+ T cells are quantified along the pseudotime cell order.(F) A representative image showing the localization of PD1+GITR+CD8+ T cells (arrow heads) in CRC tissues using multiplex immunohistochemistry (left, scale bar: 100 µm). The inset illustrates higher resolution images (right, scale bar: 25 µm). PanCK (orange) is tumor cell marker, and white depicts merged green (PD1) and purple (GITR).(G) Quantitation of PD1+, GITR+, and GITR+PD1+ expression in tumor-infiltrated CD8+ T cells. Each dot represents one tumor sample. Regression analysis showing PD1+ and GITR+CD8+ T cells numbers per mm2 (top). p value was calculated automatically by GraphPad software in linear regression module. Box and whiskers plot represents the frequency of expression of PD1 and GITR in CD8+ TILs (bottom). p values were calculated using one-way ANOVA, ***p < 0.001, ****p < 0.0001.

Fig 5: Characterization of Ad-Cab. (A) Graphical representation of the IgGA Fc-fusion protein; the cross-isotype Fc is made up of the CH chains 2 and 3 of an IgA2 (purple) and IgG1 (orange) attached to an PD-1 ectodomain (green) via a glycine linker. The IgGA Fc employs effector mechanism of both an IgG1 and IgA2. (B) Schematic representation of oncolytic adenovirus 5/3 delta 24 (Ad5/3 Δ24) constructs with modifications in the E1, E3 and fiber regions. Black inverted triangles represent deletions. Both unarmed Ad5/3 Δ24 (Unarmed) and IgGA PD-L1 Ad-5/3 △24 (Ad-Cab) have a 24 base-pair deletion in the E1 region, leading to conditionally replicate in Rb-deficient cells, and a serotype 5 fiber knob with serotype 3 knob. The IgGA PD-L1 fusion protein cassette consisted of CMV promoter and enhancer and was cloned into the gp19k+71 .k region. (C) Quantification of IgGA Fc-fusion proteins over time. A549 cells were infected with 100 MOI of Ad-Cab and Unarmed virus and supernatants were collected at different indicated time points. IgGA Fc-fusion proteins were purified, and concentration was assessed by measuring absorbance at 280 nm. (D) Competitive assay between Atezolizumab and Ad-Cab. A549 cells were incubated with different concentrations of purified IgGA Fc-fusion proteins from Ad-Cab and followed by addition of 10 µg/mL Atezolizumab. Atezolizumab binding was then analyzed using an PE-labeled antihuman IgG not recognizing IgGA Fc-fusion proteins. (E) Coincubation of monocyte-derived dendritic cells with allogenic CFSE stained T cells, at a 1:10 ratio, in the presence of 1 µg/mL of Atezolizumab or isolated IgGA Fc-fusion peptide. CFSE was then measure from CD3+ CD+ 8 T cells and the expansion index was calculated. CH, constant heavy; CMV, cytomegalovirus.