Fig 2: Aged Gal9KO mice develop spontaneous autoimmunity.(A) Spleen weight of WT (black) or Gal9KO (red) mice aged >8 months. (B) Representative plots of GC B cells in the spleen of aged mice, as indicated. (C) Summary proportion of GC B cells, as in (B). (D) Representative plots of antibody secreting cells (ASC) in the spleen of aged mice. (E) Summary proportion of ASC, as in (D). (F) Proportion of ASC lacking B220 expression. (G) Representative images of HEp-2 cells stained with sera from WT (top) or Gal9KO (bottom) mice. (H) Summary mean fluorescence intensity (MFI) of IgM (left) and IgG (right) of HEp-2 staining as in (G). (I) Anti-dsDNA specific IgG titers. (J) nti-glomerulus membrane extract (GME) specific IgG titers. (K) IgG-containing immune complexes (IC) titers. (L) Proteinuria, determined by protein secretion into the urine. Data are representative of 10–20 biological replicates over at least three independent experiments. Statistical significance was assessed by Mann–Whitney **p=0.01, ****p<0.0001.

Fig 3: Selection of broadly reactive IgG is regulated differently from strain-specific IgG.a B220+IgM-IgD-Dump- B cells were obtained from spleen of the mice immunized with inactivated A/Narita/1/2009 (Vaccine, white bars; n = 3) and from MLN in the A/Narita/1/2009 infected mice (Infection, black bars; n = 3). Binding to A/Narita/1/2009-HA and/or A/PR/8/1934-HA was determined by flow cytometry analysis. b The data indicate binding to HA in flow cytometry analysis (left) and the number of single or dual-binding B cells (right) in spleen (white bars) or MLN (black bars) from the mice infected with A/Narita/1/2009 (n = 5). c Narita-HA binding B cells (2000 cells each) were isolated from vaccinated mice (spleen, white bar; n = 3) and infected mice (MLN, black bar; N = 3). The V-D-J combinations are shown by connected color bands between V and D (left), V and J (middle), and D and J (right). d Vh gene usage of A/Narita/1/2009-HA binding B cells in spleen from three vaccinated mice (Vaccine n = 3) or MLN (Infection n = 2) from two infected mice with A/Narita/1/2009. Total RNA isolated from sorted A/Narita/1/2009-HA binding B cells served as the template for cDNA synthesis followed by PCR to amplify IgG variable region genes. Amplified IgG gene libraries were sequenced by MiSeq. The IgG sequences were analyzed with the ImMunoGeneTics (IMGT) HighV-QUEST. e Seven hundred twenty HA probe+ B cells were single sorted and cultured on NB21.2D9 feeder cells with IL-4 in individual wells. After 20 days of culture, 303 IgG-producing cells were obtained. The binding of antibodies produced by these cells to A/Narita/1/2009-HA, A/PR/8/1934-HA, A/H1N1pdm09 derived LAH (blue), or rHA2 (red) was measured by ELISA. Finally, reliable binding and VH genetic information were obtained for 101 clones. Black circles indicate clones showing no binding to LAH or rHA2. Y-axis represents the ratio of the OD450 value in the sample vs the OD450 value obtained with IgG [anti-Narita-HA (1 µg/ml), anti-PR8-HA (1 µg/ml), anti-LAH (1 µg/ml), and rHA-2 (3 µg/ml)]. f Amino acid sequences encoded by Vh genes in single or dual-HA binding B cell clones in the region from CDR1 to CDR3 are shown with germline amino acid sequence (unhighlighted). Red boxes indicated CDR regions. The positions where amino acid substitutions occur are indicated by the red font. g Number of amino-acid mutations in the region extending from CDR1 to CDR3 of single or dual-binding B cell clones (single, blue dots; n = 18, dual, red dots; n = 31) as determined by referring to the germline sequences of the corresponding Vh in the IMGT database. *p < 0.05, **p < 0.01 by unpaired, two-tailed t-test (Vaccine group with Infection group in a and c, Spleen group with MLN group in b, Single group with Dual group in g). All error bars represent SEM.

Fig 4: GC function is required for cross-strain protective humoral responses.a Top panel indicates the design of the BALF transfer experiment for protective antibody in Bcl6-deficient mice. Mice intranasally administered with BALF or serum (60 µg/ml anti-Narita IgG) from WT (white), Bcl6?T (black) or Bcl6?B (red) mice infected with A/Narita/1/2009 were challenged intranasally with a lethal dose of A/Narita/1/2009 (left) (BALF WT n = 3, BALF Bcl6?T n = 4, Bcl6?B n = 4, Serum WT n = 3, Bcl6?T n = 5, Bcl6?B n = 6) or A/PR/8/1934 (right) (WT n = 8, Bcl6?T n = 10, Bcl6?B n = 4, Serum WT n = 4, Bcl6?T n = 5, Bcl6?B n = 6). The data indicate body weight change. Missing symbols mean that the mice were euthanized, as described in Fig. 1b. b A/H1N1pdm09-HA- and A/PR/8/1934HA-specific IgG1 and IgG2 titers in BALF from WT (white bars), Bcl6?T (black bars), and Bcl6?B (red bars) mice (n = 7 each) at 14 days post-infection with A/Narita/1/2009 were measured by ELISA. c The data indicate single or dual-binding B cells in the B220+IgM-IgD-Dump- population from MLN of WT (white bars, n = 4), Bcl6?T (black bars, n = 5), and Bcl6?B (red bars, n = 7) mice. UI represents unimmunized. Bars represent mean; *,#p < 0.05, **,##p < 0.01 by unpaired, two-tailed t-test (WT group with other groups respectively). All error bars represent SEM.

Fig 5: PD-1 Blockade at the Peak of Viral Load Results in Reduced Spleen Sizes and Pan-lymphopenia(A) Gross images of spleens from control and anti-PD-L1-treated mice on 22 dpi.(B) Splenocyte counts of control and anti-PD-L1-treated mice on 22 dpi. n > 8 mice per group, shown by mean and SD.(C) H&E staining of spleen sections from control and day 8–22 anti-PD-L1-treated mice on 22 dpi. Scale bars, 200 µm. Representative images of 6 mice per group are shown.(D) Expression of CD44 and LCMV-gp33-specific TCR of total splenocytes and expression of TCF-1 and TIM-3 by LCMV-gp33-specific CD8 T cells in control and anti-PD-L1-treated mice on 22 dpi.(E) Statistical analysis of frequencies and absolute numbers of the indicated cell populations pooled from 2 experiments. n = 4–6 mice per group.(F) Expression of IFN-? and TNF-a by splenocytes from LCMV-c13-infected control and anti-PD-L1-treated B6 mice following stimulation with the gp276–284 peptide. Representative data from 2 experiments are shown with mean ± SD; n = 4.(G) Expression of Ki-67 by gp33-specific CD8 T cells.(H) Frequencies of CD45.2 PD-1+ donor CD8 T cells in PBMCs of CD45.1-recipient mice that received PD-1+ CD8 T cells from LCMV-c13-infected anti-PD-L1-treated B6 mice and subsequently challenged with LCMV-c13. Data are pooled from two experiments.(I) Expression of Fas and GL7 expression in B220+ splenocytes from control and anti-PD-L1-treated mice. Data from 2 experiments are shown with mean and SD.(J) Anti-LCMV IgG2c antibody titers on 22 dpi.See also Figure S2.