KSHV induces immunoglobulin rearrangements in mature B lymphocytes

<div><p>Kaposi sarcoma herpesvirus (KSHV/HHV-8) is a B cell tropic human pathogen, which is present <i>in vivo</i> in monotypic immunoglobulin λ (Igλ) light chain but polyclonal B cells. In the current study, we use cell sorting to infect specific B cell lineages from human tonsil specimens in order to examine the immunophenotypic alterations associated with KSHV infection. We describe IL-6 dependent maturation of naïve B lymphocytes in response to KSHV infection and determine that the Igλ monotypic bias of KSHV infection <i>in vivo</i> is due to viral induction of BCR revision. Infection of immunoglobulin κ (Igκ) naïve B cells induces expression of Igλ and isotypic inclusion, with eventual loss of Igκ. We show that this phenotypic shift occurs via re-induction of Rag-mediated V(D)J recombination. These data explain the selective presence of KSHV in Igλ B cells <i>in vivo</i> and provide the first evidence that a human pathogen can manipulate the molecular mechanisms responsible for immunoglobulin diversity.</p></div

IL-6 signaling does not affect BCR revision, but controls MZL phenotype in KSHV-infected cultures.

<p>(A) Naïve B lymphocyte cultures were infected with KSHV or Mock infected and treated with PBS (Control), IL6 neutralizing antibody at 200ng/ml or gp130 neutralizing antibody at 2μg/ml. At 6 days post-infection, cells were analyzed by FCM and subsets were gated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006967#ppat.1006967.g002" target="_blank">Fig 2D</a>. Results were verified in two independent experiments with two tonsil specimens. (B) Aggregate data for 10 individual observations from 6 independent experiments correlating the induction of IL6 (KSHV/Mock) from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006967#ppat.1006967.g002" target="_blank">Fig 2A</a> with the MZL (IgD+CD27+) population in the same sample at the same timepoint. Log regression was performed in R software using least means method (<i>r</i>² = 0.82, p = 0.0003) and gray shading represents a 95% confidence interval. (C) Mock or KSHV infected naïve B lymphocytes were treated with IL-6 or gp130 antibodies and analyzed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006967#ppat.1006967.g001" target="_blank">Fig 1B</a>. Plots shown are from a singlet/viable/CD19+CD38low gating hierarchy in a representative experiment and results were verified in two independent experiments with two tonsil specimens.</p

KSHV infection of naïve B lymphocytes recapitulates features of MCD.

<p>(A) Concentrations of human IL-6 in culture supernatants were determined in mock and KSHV-infected naïve B lymphocyte cultures at timepoints between 3 and 10 days post-infection by bead-based immunoassay (BD Cytokine Bead Array). Data represent 9 independent experiments with 4 tonsil specimens. p<0.0001 for aggregate data comparing Mock vs. KSHV. p = 0.003 for Mock vs. KSHV at 5 days post-infection and p = 0.004 for Mock vs. KSHV at 6 days post-infection. Primary naïve B lymphocytes were magnetically sorted from total tonsil lymphocytes and infected with KSHV or Mock infected. At timepoints between 3 and 13 days post-infection 2e5 cells were removed from each culture and analyzed by FCM (B) representative plots gated on single, CD19+ population from a representative experiment at 5 and 10 dpi. (C) Aggregate data for 6 independent experiments from 5 tonsil specimens showing the percent of each subset, which expressed both IgD and CD27 at indicated timepoints post-infection. Additional linear mixed model regression on each independent experiment followed by ANOVA (Type II Wald F tests with Kenward-Roger df) analysis revealed a significant effect of KSHV infection (F = 16.5, p = 0.0005) and both GFP negative (p<0.0001) and GFP positive (p = 0.02) populations were significantly different from Mock based on post-hoc Tukey test. <b>(</b>D) FCM plots gated on CD19+GFP- or CD19+/GFP+ populations from a representative experiment at 3, 7 and 10 dpi. (E) Aggregate data for 11 experiments from 8 individual tonsil specimens showing the percent of the CD19+ population with each light chain phenotype (Igκ+, Igκ+Igλ+ and Igλ) over the timecourse of infection. Linear regression was performed in R software using least means method and gray shading represents a 95% confidence interval. Additional linear mixed model regression on each independent experiment followed by ANOVA (Type II Wald F tests with Kenward-Roger df) analysis revealed significant effects of KSHV infection for each light chain immunophenotype (for Igκ: F = 147.7, p = 2.3E-15; for Igκ+Igλ: F = 52.4, p = 6.3E-9; for Igλ: F = 16.5, p = 0.0002).</p

KSHV-infected Igκ+ lymphocytes become Igλ+ via an isotypically included intermediate.

<p>Primary naïve B lymphocytes were magnetically sorted from total tonsil lymphocytes, then subsequently flow sorted based on light chain expression and infected with KSHV or mock-infected. At 3, 5 and 7 days post-infection 2e5 cells were removed from each culture and analyzed by FCM for expression of Igκ and Igλ. (A) FCM plots gated as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006967#ppat.1006967.s002" target="_blank">S2A Fig</a> from a representative experiment. (B) Aggregate data for 6 experiments from 6 individual tonsil specimens showing the percent of the single, viable, CD19+ population with each light chain phenotype (Igκ+, Igκ+Igλ+ and Igλ+) based on gates shown in (A) over the time course of infection. Linear regression on the aggregate data was performed in R software using least means method and gray shading represents a 95% confidence interval. Additional linear mixed model regression on each independent experiment followed by ANOVA (Type II Wald F test with Kenward-Roger df) analysis revealed highly significant and dominant effects of KSHV infection for each light chain immunophenotype (for Igκ: F = 58.05, p = 5.8E-8; for Igκ+Igλ: F = 27.95, p = 1.8E-5; for Igλ: F = 35.06, p = 3.5E-6). (C) Aggregate data as in (b) for KSHV-infected samples only separating GFP+ and GFP- events. Biexponential regression was performed in R software using least means method and gray shading represents a 95% confidence interval.</p

Validation of V(D)J recombination in KSHV infected cultures.

<p>(A) Primary naïve B lymphocytes were magnetically sorted from total tonsil lymphocytes, then subsequently flow sorted based on light chain expression and infected with KSHV or mock-infected. At 4 days post-infection total genomic DNA was harvested and BIOMED2 primers were used to amplify functional V-J rearrangements by PCR. (B) Mock Igλ+ or KSHV Igκ+ cultures were flow sorted at 7dpi for Igλ+ (Mock Control) or GFP+Igλ+ (KSHV-modified) and single cells were collected in 96-well PCR plates. RT-PCR was performed from single cell cDNA for Igλ transcripts. Data represents the percent of single cells expressing Igλ transcripts from three individual tonsil donors in three independent experiments. n≥96 for each sample. (C) KSHV Igκ+ cultures were flow sorted at 7dpi and GFP+Igκ+Igλ+ single cells were collected in 96-well PCR plates. RT-PCR was performed from single cell cDNA for both Igλ and Igκ transcripts. Data represents the number of single cells amplifying the indicated light chains or with no amplification (NA), overall n = 480 from three individual tonsil donors in two independent experiments. (D) Primary naïve B lymphocytes were KSHV or mock-infected. Total mRNA was harvested at 4 hours post-infection and RAG1 and RAG2 transcripts were amplified by nested RT-PCR. RT-PCR results were verified in 6 individual tonsil samples. (E) KSHV-modified Igλ transcripts (from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006967#ppat.1006967.g004" target="_blank">Fig 4B</a>, representing single infections from three individual tonsil donors) were sequenced and immunoglobulin lambda variable (IGLV) gene families were determined using IgBLAST (NCBI). Data displays the histogram actual frequency and Gaussian kernel density distribution for each IGLV gene family for Control (Mock Igλ+) and KSHV-modified (Igκ+ at infection, GFP+Igλ+ at sorting) sequences. Kolmogorov-Smirnov test indicates a statistically significant difference between the distributions (p<0.0001).</p

A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV.

<p>Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and >40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p<0.0001. Student’s T test revealed no significant difference between Cq values for Mock and NRT samples for any target gene. (D) Magnetically sorted naïve B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed as in (A) for GFP expression.</p

Supplementary files from Novel Richter Syndrome Xenograft Models to Study Genetic Architecture, Biology, and Therapy Responses

This file contains supplemental additional experimental procedures, relevant references and 7 supplementary figures, including: S1 "Expression of EBV in RS samples"; S2 "Schematic representation of the RS-PDX models"; S3 "Disease engraftment and distribution of RS1316 i.v. model"; S4 "Phenotypic analysis of primary and derived-RS-PDXs"; S5 "Schematic diagram of proteins and corresponding mutations"; S6 "Differentially expressed genes in primary and RS-PDX"; S7 "Schematic sketch of the main mutated genes in our RS models and core signaling pathways they belong to and play a critical role". Supplementary tables contain additional information on sequencing results performed on primary and PDX-RS models.</p

Additional file 1 of Metagenomic analysis to identify novel infectious agents in systemic anaplastic large cell lymphoma

Additional file 1: Figure S1. Schematic of the computational approach used in the metagenomic analysis of ALCL. Figure S2. Additional GATK-PathSeq analysis of tumor specimens. Figure S3. Detailed taxonomic classification of GATK-PathSeq assigned non-human reads. Figure S4. Use of read-based approach to identify viral sequences associated with ALCL. Figure S5. The kmer enrichment approach to identify pathogen reads from unmapped GATK-PathSeq non-human reads. Table S1. Quality control results for cases included in the analysis. Table S2. Demographic and immunophenotypic features of cases and controls. Table S3 Immunohistochemistry and in situ hybridization panel performed on ALCL and DLBCL tumor specimens

<i>In situ</i> hybridization for EBER-positive cells in lymph node tissue.

(A-D) Representative images from in situ hybridization assays to detect Epstein-Barr virus(EBV)-encoded small RNAs (EBER) positive cells in lymph node tissue from (A) iMCD (n = 12), (B) UCD (n = 9), (C) HHV-8-positive MCD (n = 4), and (D) EBV-associated lymphoproliferative disorder (n = 4) cases. Images are shown at 10x magnification. (E) The average number of EBER positive cells per field, across ten high power fields (HPF) at 40x for each case is presented across all cases of each diagnosis. Values are presented as mean ± standard deviation, as appropriate. P-values are derived from one-sided t-test with Bonferroni correction.</p

Summary of viral transcripts detected by VirCapSeq-VERT.

Summary of viral transcripts detected by VirCapSeq-VERT.</p