Plasmodium Infection Promotes Genomic Instability and AID-Dependent B Cell Lymphoma

SummaryChronic infection with Plasmodium falciparum was epidemiologically associated with endemic Burkitt’s lymphoma, a mature B cell cancer characterized by chromosome translocation between the c-myc oncogene and Igh, over 50 years ago. Whether infection promotes B cell lymphoma, and if so by which mechanism, remains unknown. To investigate the relationship between parasitic disease and lymphomagenesis, we used Plasmodium chabaudi (Pc) to produce chronic malaria infection in mice. Pc induces prolonged expansion of germinal centers (GCs), unique compartments in which B cells undergo rapid clonal expansion and express activation-induced cytidine deaminase (AID), a DNA mutator. GC B cells elicited during Pc infection suffer widespread DNA damage, leading to chromosome translocations. Although infection does not change the overall rate, it modifies lymphomagenesis to favor mature B cell lymphomas that are AID dependent and show chromosome translocations. Thus, malaria infection favors mature B cell cancers by eliciting protracted AID expression in GC B cells.PaperCli

RPA Accumulation during Class Switch Recombination Represents 5′–3′ DNA-End Resection during the S–G2/M Phase of the Cell Cycle

Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S–G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S–G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S–G2/M phase of the cell cycle

Mapping AID-tagged cells during development.

<p><b>A</b>. Fluorescence imaging of partially cleared AID ^cre/+ ROSA^>TOM/+ embryos reveals a signal within the head (arrows). The letter “e” indicates the location of the eye in the higher magnification images. Top and bottom panels represent embryos at dpc 11.5 and 12.5, respectively. All images display tdTomato signal, with the exception of the top left panel, which is phase contrast of the image to its right. <b>B</b>. Left: Summary dot plot with the percentages of CD45^-YFP⁺ cells in dissected brains from AID ^cre/+ ROSA^>YFP/YFP embryos at different days post conception. P2 is postnatal day 2. Each symbol represents an independent brain. n.d. is non-detectable over control. Right: Representative FACS plots of dpc 15.5 dissected brains. Numbers are the percentage of YFP⁺ cells in the shown gate. <b>C</b>. Macroscopic appearance of Lig4^-/-AID^-/- embryos (18.5 dpc) alongside controls. <b>D</b>. Table summarizing the genotypes of 75 embryos (18.5 dpc) obtained from the intercross of Lig4^+/-AID^+/- parents. Eleven litters were analyzed in total. In parenthesis is the number of embryos hemorrhagic or partially resorbed at the time of analysis. DNA from 6 additional embryos was degraded and genotyping could not be performed.</p

Papillomas in AID ^cre/+ Pten^lox/lox.

<p>Macroscopic (<b>A</b>) and histologic (<b>B</b>) appearance of papillomas in a representative 8 months old AID ^cre/+ Pten^lox/lox mouse. <b>C</b>. β-galactosidase staining of whole AID ^cre/+ ROSA^>GNZ/+ embryos at dpc 14.5 reveals a signal (blue) in the submandibular region. Similar results were obtained at dpc 15.5 (not shown).</p

AID in the adult mouse brain.

<p><b>A</b>. Immuno-histochemical detection of GFP/YFP in the brains of 8 weeks old mice. Shown is coronal section 25 (out of 71, starting from rostral). <b>B</b>. Higher magnification of the boxed areas in panel A demonstrating expression in pyramidal layer IV (left) and septal area (right). <b>C</b>. Immuno-fluorescence images of sagittal sections of AID ^cre/+ ROSA^>YFP/YFP brains showing reporter staining in scattered granule (i) and Purkinje (ii) cells in cerebellum, and granule cells in the dentate gyrus (iii). More uniform staining is observed in the antero-ventral nucleus of thalamus (iv) and cortex layer IV (v).</p

AID during development.

<p><b>A</b>. Representative FACS plots of whole embryos revealing YFP ⁺ CD45^- cells starting at 10.5 dpc. Numbers are the percentage of YFP⁺ cells in the shown gate. <b>B</b>. Summary dot plot with the percentage of YFP ⁺ CD45^- cells in AID^cre/+ ROSA^>YFP/YFP embryos at the indicated days post conception (dpc). Each triangle represents an individual embryo. n.d. is non-detectable over control. <b>C</b>. Representative FACS plots showing AID^GFP+ cells at dpc 11.5. <b>D</b>. Summary dot plot with the percentage of GFP⁺ cells in AID^GFP embryos at the indicated developmental stages (days post conception). Each triangle represents an individual embryo. For dpc 9.5-12.5, cells from the whole embryo were analyzed; for dpc 13.5-16.5 analysis was limited to the upper body half. n.d. is non-detectable over wild type control. <b>E</b>. Ethidium bromide stained agarose gels with RT-PCR products obtained from sorted AID^GFP+ germinal center B cells from Peyer’s Patches (positive control). Numbers represent cell equivalents, w is water control. GAPDH control is shown alongside. <b>F</b>. RT-PCR products from sorted AID^GFP+ cells at 11.5 dpc from three independent experiments (lanes 1, 2, and 3). Triangles represent increasing amounts of template cDNA. Cell equivalents for the AID nested PCR were: 10, 20 (experiment 1); 5, 10, 50 (experiment 2); 1, 2, 10 (experiment 3). + is positive control and w is water. Cell equivalents for GAPDH were 10, 10, and 5 for experiments 1, 2, and 3 respectively.</p

Fate mapping identifies known AID-expressing cells.

<p><b>A</b>. Schematic representation of the genetic elements used for fate mapping. Cre-mediated excision of a loxP-flanked transcriptional stop allows for reporter expression in AID-expressing cells and their descendants. <b>B</b>. YFP expression in Peyer’s Patches by immuno-fluorescent staining (top) and flow cytometry (bottom). YFP⁺ cells are positive for the B cell marker CD19. Numbers are the percentage of cells within the shown gate. <b>C</b>. Flow cytometry identifies YFP ⁺ CD138⁺ post-germinal center plasma cells in spleen and bone marrow. <b>D</b>. YFP ⁺ CD3⁺ T cells are identified in 8 months old mice. <b>E</b>. Absence of YFP⁺ epithelial cells after immuno-staining of prostate or small intestine. The YFP⁺ cells in the small intestine are B lymphocytes and plasma cells, which are normally present in the lamina propria located beneath the mucosal epithelium.</p

Mechanism of DNA resection during intrachromosomal recombination and immunoglobulin class switching.

DNA double-strand breaks (DSBs) are byproducts of normal cellular metabolism and obligate intermediates in antigen receptor diversification reactions. These lesions are potentially dangerous because they can lead to deletion of genetic material or chromosome translocation. The chromatin-binding protein 53BP1 and the histone variant H2AX are required for efficient class switch (CSR) and V(D)J recombination in part because they protect DNA ends from resection and thereby favor nonhomologous end joining (NHEJ). Here, we examine the mechanism of DNA end resection in primary B cells. We find that resection depends on both CtBP-interacting protein (CtIP, Rbbp8) and exonuclease 1 (Exo1). Inhibition of CtIP partially rescues the CSR defect in 53BP1- and H2AX-deficient lymphocytes, as does interference with the RecQ helicases Bloom (Blm) and Werner (Wrn). We conclude that CtIP, Exo1, and RecQ helicases contribute to the metabolism of DNA ends during DSB repair in B lymphocytes and that minimizing resection favors efficient CSR

Potentiating adoptive cell therapy using synthetic IL-9 receptors

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rβ-ECD-IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells,&nbsp;o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours