Fig 2: (A) Animal immunization and sample collection scheme. Macaque groups were immunized with the indicated Ad vectors all containing the rhFLSC transgene. O = oral, IN = intranasal, IT = intratracheal and IM = intramuscular. (B,C) The percentages of the combined levels of all three cytokines (IFNγ, TNFα and IL-2) that were expressed by memory CD4+ (panel B) and CD8+ (panel C) T-cells in PBMC from the rhesus macaques immunized with the indicated vectors are shown over the course of immunization. (D,E) The percentages of IFNγ cytokine-producing memory CD4+ and CD8+ T-cells in PBMC of rhesus macaques at wk 38 post-immunization. p values were obtained using one-way ANOVA with Tukey’s multiple comparisons Post Hoc test and for (E) were corrected by the method of Sidak for all the possible pairwise comparisons.

Fig 3: Virological and immunological features of the Lambda variant(A) Pseudovirus assay. HIV-1-based reporter viruses pseudotyped with SARS-CoV-2 S proteins of the parental (B.1), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Epsilon (B.1.427), and Lambda (C.37) variants and the Lambda + N246-253RSYLTPGD derivative were prepared as described in STAR Methods. The mutations in each variant are listed in Figure S1C. The pseudoviruses were inoculated in HOS-ACE2/TMPRSS2 cells at 1,000 ng HIV-1 p24 antigen, and the percentages of infectivity compared to that of the virus pseudotyped with parental S are shown.(B) Neutralization assay. A neutralization assay was performed using the pseudoviruses with the S proteins of the parental, Lambda, and Lambda + N246-253RSYLTPGD and 18 BNT162b2-vaccinated sera as described in STAR Methods. The raw data are shown in Figure S2B. The number in the panel indicates the fold change in neutralization resistance of Lambda S to the parental S or the Lambda + N246-253RSYLTPGD derivative.(C and D) Structural insights of the mutations in Lambda S. (C) Overlaid overviews of the crystal structure of SARS-CoV-2 S (PDB: 6ZGE, white) (Wrobel et al., 2020) and a homology model of Lambda S (brown) are shown. The mutated residues in the Lambda S and the regions in the NTD and RBD are indicated in red and blue. The squared regions are enlarged in (D). Mutated residues in the NTD (left) and RBD (right) of Lambda S. The residues in parental S and the Lambda S are indicated in red and black.(E) Pseudovirus assay. HIV-1-based reporter viruses pseudotyped with SARS-CoV-2 S proteins bearing respective mutations of the Lambda variant in parental S (G75V, T76I, GT75-76VI, RSYLTPGD246-253N, L452Q, F490S, L452Q/F490S, or T859N) as well as the parental S were prepared. The pseudoviruses were inoculated into HOS-ACE2/TMPRSS2 cells at 1,000 ng HIV-1 p24 antigen, and the percentages of infectivity compared to that of the virus pseudotyped with parental S are shown.(F) Binding affinity of the SARS-CoV-2 S RBD (residues 336-528) to ACE2 by yeast surface display. The KD values of the binding of the SARS-CoV-2 S RBD expressed on yeast to soluble ACE2 are shown.(G) CTL assay. HLA-A24+ CTL lines established from 6 BNT162b2-vaccinated donors were stimulated with 1 nM NF9 peptide or its derivatives, NF9-L452Q (NYNYQYRLF) or NF9-L452R (NYNYRYRLF). The percentage of IFN-?+ cells in CD8+ T cells (bottom, n = 6) is shown. Representative fluorescence-activated cell sorting (FACS) plots showing intracellular expression of IFN-? in the CD8+ T cell subset of a vaccinated donor (top, donor GV36-2) are shown in Figure S2H.(H) Neutralization assay. A neutralization assay was performed using the pseudoviruses used in Figure 2B and 18 BNT162b2-vaccinated sera, as described in STAR Methods. The raw data are shown in Figure S2B. The number in the panel indicates the fold change in neutralization resistance to the parental S.(I–L) Effect of monoclonal antibodies. The mean florescence intensity (MFI) of the surface S proteins stained with NTD-targeting NAb clone 4A8 (151 ng/mL–0.21 ng/mL) (Chi et al., 2020) (I) and EAb clone COV2-2490 (1 µg/mL–0.10 µg/mL) (Liu et al., 2021c) (K) antibodies. (J) Antiviral effect of the NTD-targeting NAb clone 4A8 antibody. (L) Enhancing effect of the EAb clone COV2-2490 antibody. The percentages of infectivity compared to that of the virus without antibodies are shown.Assays were performed in triplicate (except for H) or quadruplicate (H), and the mean is shown with the SD.In (A), (E), (F), (I), and (K), statistically significant differences (*, p < 0.05) versus parental S were determined by Student’s t test. In (A) and (E), vertical dashed lines indicate 100% (the value of parental S).In (B) and (H), statistically significant differences were determined by the Wilcoxon matched-pairs signed rank test. The p values are indicated in the figure.In (G), statistically significant differences (*p < 0.05) versus the NF9 peptide were determined by the Wilcoxon matched-pairs signed rank test.In (I) and (K), vertical dashed lines indicate the MFIs of mock-transfected cells. In (J), statistically significant differences (*p < 0.05) versus the value without the antibody were determined by Student’s t test. NS, no statistical significance. In (L), statistically significant differences versus the value without the antibody (*p < 0.05) and the parental S (#p < 0.05) were determined by Student’s t test.See also Figure S2 and Tables S4 and S5.

Fig 4: IL-4–induced STAT6 signaling inhibits CD39 expression.(A) Purified total T cells from human peripheral blood were activated for 4 days by anti-CD3/CD28 Dynabeads in the presence or absence of IL-4 (20 ng/mL). CD39 expression was compared by flow cytometric analysis. Left are representative contour plots and right are summary results. (B) Total T cells were treated as described in A and collected for ENTPD1 transcript quantification. (C) PBMCs were activated for 4 days with soluble anti-CD3 Ab (2 µg/mL) in the presence or absence of IL-4. Results on CD39 expression on gated CD19+ B cells are shown as representative histograms (left) and summary data (right). (D) Mice were implanted with B16 melanoma cells. Splenocytes and TILs were harvested on day 26 and expression of IL-4R on indicated CD4 and CD8 T cell subsets was determined. Contour plots of CD39 and TIM3 expression (left) and histograms of IL-4R expression (right) are representative of 2 experiments. Data in B are shown as mean ± SEM and compared by 2-tailed paired Student’s t test in A–C.

Fig 5: T cells from FAP patients present migration defects.(A) Transmigration through transwell filters was assessed for T cells from FAP patients (FAP) and matched healthy donors (HD). Bars represent mean + SEM (six individual pairs) of specific migration of CD8 and CD4 T cells. Statistical differences were calculated by paired t test. ***P < 0.001. (B to F) T cells from FAP patients and matched HDs were reactivated after thawing. (B) Bar graphs represent mean + SEM (three individual pairs) of specific transwell migration of CD8 and CD4 T cells through HUVEC monolayers. Statistical differences were calculated by paired t test. *P < 0.05. (C to F) CD8 T cells were sorted after thawing and were assessed for their migration in 3D collagen gels (C) or in microchannels (D to F). Boxes illustrate 10th to 90th percentiles of values, and whiskers represent the range of values. Statistical differences were calculated by Mann-Whitney unpaired test. ***P < 0.001 and ****P < 0.0001; ns, not significant. (C) CD8 T cell migration in 3D collagen matrices was recorded by time-lapse video microscopy. Mean speed of migrating cells from four individual pairs is shown. (D to F) T cell migration through collagen type I–coated (D) or fibronectin-coated (E and F) microchannels was assessed for CD8 T cells from three and five matched individuals, respectively. (F) Left panel shows sequential images of Hoechst-labeled CD8 T cells from a representative individual pair (20× objective). Scale bars, 10 μm. Right panels show video microscopy frames (63× objective) (movie S1). Insets zoom in the front edge’s areas of migrating cells displaying cell lamellipodium (bracket) or lobe-shaped protrusions or blebs (empty arrowheads).