Med1 deficiency alters the somatic hypermutation spectrum in murine B cells.

The Mediator complex is known to orchestrate transcription. Here we show that B cell conditional deficient mice for the Med1 subunit display robust somatic hypermutation. Nevertheless, the mutation frequency at A residues is decreased and the expected A/T ratio is abolished, implicating Mediator in the second phase of somatic hypermutation.

Robust immunoglobulin class switch recombination and end joining in Parp9-deficient mice.

To mount highly specific and adapted immune responses, B lymphocytes assemble and diversify their antibody repertoire through mechanisms involving the formation of programmed DNA damage. Immunoglobulin class switch recombination (CSR) is triggered by DNA lesions induced by activation-induced cytidine deaminase, which are processed to double-stranded DNA break (DSB) intermediates. These DSBs activate the cellular DNA damage response and enroll numerous DNA repair factors, involving poly(ADP-ribose) polymerases Parp1, Parp2, and Parp3 to promote appropriate DNA repair and efficient long-range recombination. The macroParp Parp9, which is overexpressed in certain lymphomas, has been recently implicated in DSB repair, acting together with Parp1. Here, we examine the contribution of Parp9 to the resolution of physiological DSBs incurred during V(D)J recombination and CSR by generating Parp9-/- mice. We find that Parp9-deficient mice are viable, fertile, and do not show any overt phenotype. Moreover, we find that Parp9 is dispensable for B-cell development. Finally, we show that CSR and DNA end-joining are robust in the absence of Parp9, indicating that Parp9 is not essential in vivo to achieve physiological DSB repair, or that strong compensatory mechanisms exist.

Inflammation enhances the vaccination potential of CD40-activated B cells in mice.

Vaccination with antigen-pulsed CD40-activated B (CD40-B) cells can efficiently lead to the in vivo differentiation of naive CD8+ T cells into fully functional effectors. In contrast to bone marrow-derived dendritic cell (BMDC) vaccination, CD40-B cell priming does not allow for memory CD8+ T-cell generation but the reason for this deficiency is unknown. Here, we show that compared to BMDCs, murine CD40-B cells induce lower expression of several genes regulated by T-cell receptor signaling, costimulation, and inflammation (signals 1-3) in mouse T cells. The reduced provision of signals 1 and 2 by CD40-B cells can be explained by a reduction in the quality and duration of the interactions with naive CD8+ T cells as compared to BMDCs. Furthermore, CD40-B cells produce less inflammatory mediators, such as IL-12 and type I interferon, and increasing inflammation by coadministration of polyriboinosinic-polyribocytidylic acid with CD40-B-cell immunization allowed for the generation of long-lived and functional CD8+ memory T cells. In conclusion, it is possible to manipulate CD40-B-cell vaccination to promote the formation of long-lived functional CD8+ memory T cells, a key step before translating the use of CD40-B cells for therapeutic vaccination.

Parp3 negatively regulates immunoglobulin class switch recombination.

To generate highly specific and adapted immune responses, B cells diversify their antibody repertoire through mechanisms involving the generation of programmed DNA damage. Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by the recruitment of activation-induced cytidine deaminase (AID) to immunoglobulin loci and by the subsequent generation of DNA lesions, which are differentially processed to mutations during SHM or to double-stranded DNA break intermediates during CSR. The latter activate the DNA damage response and mobilize multiple DNA repair factors, including Parp1 and Parp2, to promote DNA repair and long-range recombination. We examined the contribution of Parp3 in CSR and SHM. We find that deficiency in Parp3 results in enhanced CSR, while SHM remains unaffected. Mechanistically, this is due to increased occupancy of AID at the donor (Sµ) switch region. We also find evidence of increased levels of DNA damage at switch region junctions and a bias towards alternative end joining in the absence of Parp3. We propose that Parp3 plays a CSR-specific role by controlling AID levels at switch regions during CSR.