Fig 2: Mild COVID-19 Induces a Sustained Enrichment of RBD-Specific IgG+ Memory B Cells(A) Representative gating of live CD3–CD14–CD16– cells for SARS-CoV-2 RBD-specific cells (RBD tetramer+decoy tetramer–) from CoV2+ and HC PBMCs at V1 and V2.(B) Number of RBD-specific B cells (RBD tetramer+decoy tetramer–CD20+) per 1 × 106 PBMCs.(C) Representative flow cytometry plots and number of RBD-specific PBs (RBD tetramer+decoy tetramer-CD20-CD138hi) (na = could not be calculated because all values 0).(D) Representative gating of RBD-specific B cells for naive B cells (CD21+CD27-) and MBCs (CD21+CD27+/CD21–CD27+/CD21–CD27– populations outlined in green).(E) Proportion of RBD-specific B cells that are naive (CD21+CD27-), classical MBCs (CD21+CD27+), or activated MBCs (CD21-CD27+/-), statistics for the proportion that are MBCs.(F) Number of RBD-specific MBCs (classical and activated).(G) Representative gating of RBD-specific MBCs for BCR isotype (IgD, IgM, IgA, and IgG) expression.(H) Proportion of RBD-specific MBCs expressing the BCR isotypes IgD, IgM, IgA, and IgG. Statistics are for the proportion that are IgG+.(I) Number of RBD-specific IgG+ MBCs.(J) Representative gating of RBD-specific MBCs for T-bet expression and number of RBD-specific T-bet+ MBCs.Statistics for unpaired data determined by 2-tailed Mann-Whitney tests and, for paired data, by 2-tailed Wilcoxon signed-rank tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value < 0.04. Error bars represent mean and SD. Data from 2 experiments per visit. See also Figure S5.

Fig 3: In vivo consequences of cis versus trans CD137 costimulation.a, schematic layout of the experiments in b–g. A mixture of 1:1 ratio of in vitro cis-costimulated (CD45.1) and trans-co-stimulated (CD45.2) CD8+ T cells from C57 mice were adoptively transferred upon intravenous injection (i.v) into Rag1-/- congenic recipients. b, frequency of CD45.1 and CD45.2 of CD3+CD8+ T cells in the blood on days 6, 12 and 20 post-adoptive transfer (n = 10). c, frequency of CD45.1 and CD45.2 of CD3+CD8+ T cells in the liver, spleen, lymph nodes and bone marrow on day 20 post-adoptive transfer (n = 10). d, intranuclear staining of phosphorylated gH2AX on CD45.1 and CD45.2 FACS-gated CD3+CD8+ in the liver, spleen, lymph nodes (LN) and bone marrow (BM) on day 20 post-adoptive cell transfer (n = 10). Data show gMFI. Each dot represents a single mouse. Expression of T-bet (e) and Eomes (f) and T-bet:Eomes ratio (g) in CD45.1 and CD45.2 FACS-gated CD3+CD8+ in the bone marrow on day 20 post-adoptive cell transfer (n = 10). Data show geometric mean fluorescence intensity (gMFI). Each dot represents a single mouse. h, experimental layout of the experiment in i. A mixture of 1:1 ratio of cis-costimulated (OT-1 CD45.1+) and trans-co-stimulated (OT-1 CD45.2+) CD8+ T cells were adoptively transferred into subcutaneous (s.c) implanted B16.OVA tumor-bearing Rag1-/- recipient mice. i, relative frequency of OT-1 CD45.1+ and OT-1 CD45.2+ CD8+ T cells in the tumor and draining lymph nodes (dLN) on day 16 post-adoptive transfer (n = 10). Data are given as mean ± s.e.m. Statistical significance was determined with paired t test (two-sided). Source data are provided as Source Data file.

Fig 4: In vitro IL-27 stimulation increased expression of CD69 and T-bet in CD4 and CD8 T cellsPBMCs from PLWH (HIV+, n = 17) and healthy controls (HC, n = 17) were cultured in the absence (opened symbols) or presence (closed symbols) of rhIL-27 (100 ng/mL) overnight. Flow cytometry analysis was performed for the expression of: (A) CD69; (B) T-bet, GATA3, and Foxp3+CD25+; (C) TIGIT, Tim3 and LAG3 in CD4 and CD8 T cells. Expressions of the markers are expressed as frequencies of the CD4 and CD8 T cells. The graphs are represented by box and whisker showing the median value with first and third quartiles in the box, with whiskers extending to the minimum and maximum values. Comparisons between the media and IL-27 stimulation were performed by non-parametric Wilcoxon test. Comparisons between the groups were performed by non-parametric Mann–Whitney test. p value <0.05 was considered significant.

Fig 5: iNKT cells from TN and C57BL/6 mice show similar influence of tissue microenvironment on NKT1, NKT2, and NKT17 subsets A–FLymphocytes from the indicated tissues of C57BL/6 and Vα14 mice were stained with anti‐CD3 and CD1d‐(PBS57)‐tetramer, before they were fixed, permeabilized, and stained with antibodies to T‐bet, RORγt, and PLZF. Results shown are gated on CD3+CD1d‐tetramer+ cells.GThe percentage of CD3+ CD1d‐tetramer+ iNKT cells in each organ that stained positively for PLZF, T‐bet, and RORγt are shown. **P < 0.01, Mann–Whitney test. Error bars are SD.Data information: Results shown are representative of three independent experiments where n = 3 biological replicates.