Fig 1: Human Intestinal ?d Cells and the Selective Impact on Them of BTNL3 and BTNL8 Co-expression, Related to Figure 7(A) FACS-sorted ?d T cells harvested from human intestinal tissue were analyzed by deep sequencing for TCR V? chain usage. (B) Schematic illustrating the murine and human Btnl2/BTNL2 and Btnl9/BTNL9 loci, adapted from the NCBI gene viewer. (C) Conventional RT-PCR analysis of BTN3A2, BTNL3 and BTNL8 expression in the indicated tissues. (D) Conventional RT-PCR analysis of BTN3A1, BTNL3, BTNL8, EPCAM and TCR V?2/3/4 expression in the indicated samples. (E) Cell surface expression of FLAG-BTNL3, FLAG-BTNL8S or FLAG-BTNL8 co-transfected in HEK293 cells with the indicated constructs. Histogram overlays show the expression of each BTNL after gating on GFP+ cells (numbers in brackets indicate geometric mean fluorescence intensity, gMFI). (F) Schematic illustrating the method of human intestinal tissue-resident lymphocytes isolation and co-culture with HEK293 transductants. (1) Endoscopic biopsies recovered from ascending colon of healthy donors. (2) Washed in complete media supplemented with antibiotic. (3) 1 biopsy applied to each matrix. (4) Culture for 5-7 days in complete medium supplemented with antibiotics, IL-2 and IL-15. (5) Co-culture with HEK293 cell lines transduced with EV, L3, L8 or L3+8. (G) Cell surface CD25 expression on indicated subsets of human gut-derived lymphocytes after co-culture with EV versus L3+8-transduced HEK293 cells. (H) Gating parameters for sorting of Btnl3+8-responsive human gut-derived lymphocytes. (I) TCRV? chain usage (left) and cell surface TCR?d expression (right) in gut-derived ?d T cells (isolated from a donor unresponsive to BTNL3+8) after co-culture with EV versus L3+8-transduced HEK293 cells.
Fig 2: Cellular screening of SARS-CoV-2-specific response.a Memory CD8+ T cells (CD45RO+) from peripheral blood mononuclear cells (PBMC) of COVID-19 convalescents were seeded at 2000 cells/well in multiple wells containing irradiated allogenic PBMC, PHA, IL-2, IL-7, and IL-15 as described in Methods. The individual cultures were expanded to make up the libraries of polyclonally amplified CD8+ T cells. Then the libraries were washed and examined as to their antigen specificity by stimulating with a series of viral protein-expressing aAPC. Once being judged as positive by IFNγlevel (measured by ELISA), they were further expanded with relevant aAPC and cytokines. These libraries (cell lines) were subjected to several downstream analyses including epitope screening, cytokine profiling, and/or cytotoxicity assays, etc. b A representative result of the CD8+ T cell library assay. Libraries from a healthy donor (HC-010) or COVID-19 convalescent (CV-004) were divided into 7 groups, and each group was co-cultured with indicated aAPCs: S (n = 15 for HC-010; n = 47 for CV-004), M (n = 15 for HC-010; n = 47 for CV-004), N (n = 15 for HC-010; n = 47 for CV-004), ORF3a (n = 14 for HC-010; n = 41 for CV-004), or ORF1ab NSP6 (n = 14 for HC-010; n = 53 for CV-004) -expressing aAPCs, influenza virus and cytomegalovirus (Flu/CMV) derived peptides (n = 15 for HC-010; n = 47 for CV-004)-pulsed aAPC or aAPCs (None) (n = 15 for HC-010; n = 47 for CV-004). Each dot represents the IFNγ level of each library. The threshold of positivity was determined by mean + 3 SD of IFNγ level in the group co-cultures with aAPCs (indicated as “None”). c Heatmap representation of results from all participants (healthy controls (HC), convalescents from mild, moderate (mod), and severe (sev) disease) subjected to the library assay. The frequency of positive library in response to aAPCs (None), indicated viral protein-expressing, or influenza virus and cytomegalovirus (Flu/CMV) derived peptides-pulsed aAPC are shown. d Comparison of positive library frequency between healthy donors (HC) (open circles, n = 8) and COVID-19 convalescents (COVID-19) (filled circles, n = 20) in each group. p values were calculated by two-sided Mann–Whitney test. Data represent mean ± SD. **p = 0.0064. e Comparison of positive library frequency in response to SARS-CoV-2-M protein-expressing aAPC among indicated groups (healthy donors (HC) (n = 8), convalescents from mild severity COVID-19 (mild COVID-19) (n = 8), those from moderate severity COVID-19 (moderate COVID-19) (n = 8) and those from severe severity COVID-19 (severe COVID-19) (n = 4)). p values were calculated by two-sided Mann–Whitney test. Data represent mean ± SD. ***p = 0.0002; *p = 0.024.
Fig 3: To trigger CD8+ T and NK cell proliferation, costimulation by 4‐1BBL is superior to CD28. Proliferation of MART‐1‐specific HLA‐A*02‐restricted CD8+ T cells in the presence of peptide P26‐35 (500 ng/ml), cytokines (IL‐15 + IL‐21), and the indicated concentrations of: (A) soluble anti‐4‐1BB antibody clone 4B4‐1 (circles) or mIgG1 isotype controls (squares) (n = 5); (B) soluble anti‐4‐1BB antibody clone BBK‐2 (circles) or mIgG1 isotype controls (squares) (n = 5); (C) soluble anti‐4‐1BB antibody clone Urelumab (circles) or hIgG4 isotype controls (squares) (n = 5); and (D) soluble anti‐CD28 antibody (circles) or mIgG1 isotype control (squares) (n = 5). Proliferation of freshly purified NK cells in the presence of cytokines (IL‐15 + IL‐21), the absence (green symbols) or presence of either CD86/4‐1BBL‐expressing (red symbols) or parental (blue symbols) K562, and the indicated concentrations of: (E) soluble anti‐4‐1BB antibody clone 4B4‐1 (circles) or mIgG1 isotype controls (squares) (n = 28); (F) soluble anti‐4‐1BB antibody clone BBK‐2 (circles) or mIgG1 isotype controls (squares) (n = 11); (G) soluble anti‐4‐1BB antibody clone Urelumab (circles) or hIgG4 isotype controls (squares) (n = 15); and (H) soluble anti‐CD28 antibody (circles) or mIgG1 isotype controls (squares) (n = 10). Data is shown as the mean ± SEM. Normalization of the data and statistical analysis were performed as described in the Section Methods, 2.
Fig 4: Human CD8+ and γδ T cell proliferation and activation relies on three signals. Proliferation of CD8+ T cells in the presence of serial dilutions of peptide P26‐35, and (A) either CD86/4‐1BBL/HLA‐A*02‐expressing (red squares) or CD86/4‐1BBL‐expressing (blue circles) K562 cells and cytokines (IL‐7 + IL‐15 + IL‐21) (n = 5); (B) either CD86/4‐1BBL/HLA‐A*02‐expressing (red squares) or HLA‐A*02‐expressing (blue circles) K562 cells and cytokines (n = 5); and (C) CD86/4‐1BBL/HLA‐A*02‐expressing K562 cells, and the absence (open black squares) or presence of IL‐2 (red circles), IL‐7 (blue squares), IL‐15 (red triangles), IL‐21 (blue circles), or IL‐7 + IL‐15 + IL‐21 (red squares) (n = 5). Proliferation of γδ T cells in the presence of serial dilutions of an anti‐human TCR γδ antibody, and (D) either the absence (blue circles) or presence (red squares) of K562‐derived aAPC expressing CD86 and 4‐1BBL and cytokines (n = 6); (E) either parental (blue circles) or CD86/4‐1BBL‐expressing (red squares) K562 cells and cytokines (n = 6); and (F) K562‐derived aAPC expressing CD86 and 4‐1BBL, and either the absence (open black squares) or presence of IL‐2 (red circles), IL‐7 (blue squares), IL‐15 (red triangles), IL‐21 (blue circles), IL‐15 + IL‐21 (open red squares) or IL‐7 + IL‐15 + IL‐21 (solid red squares) (n = 6). Data is shown as the mean ± SEM. Normalization of the data and statistical analysis were performed as described in the Section Methods, 2.
Fig 5: Alarmin-induced genes and biological pathways potentially regulated by epigenetic modifications. Heatmap showing the differential expression at all time points of cytokine stimulation of (A) IFNβ and (B) IL-15 upregulated genes (log2 fold change >1, FDR ≤ 0.01) with concordant epigenomic changes (Epi+/Exp+). The number of Epi+/Exp + genes per cluster are indicated. (n = 8 samples per time point and condition). Network-based representation of enriched biological pathways identified by GSEA on Epi+/Exp + genes responding to (C) IFNβ or (D) IL-15 stimulation.
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