Affinity maturation of the humoral response is mediated by somatic hypermutation of the immunoglobulin (Ig) genes and selection of higher-affinity B cell clones. Activation-induced cytidine deaminase (AID) is the first of a complex series of proteins that introduce these point mutations into variable regions of the Ig genes. AID deaminates deoxycytidine residues in single-stranded DNA to deoxyuridines, which are then processed by DNA replication, base excision repair (BER), or mismatch repair (MMR). In germinal center B cells, MMR, BER, and other factors are diverted from their normal roles in preserving genomic integrity to increase diversity within the Ig locus. Both AID and these components of an emerging error-prone mutasome are regulated on many levels by complex mechanisms that are only beginning to be elucidated.

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-μ and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2′-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one λ5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and λ5 coimmunoprecipitated with the μ-chain in lysates of 293T cells transfected with VpreB, λ5, and μ, indicating that VpreB, λ5, and μ-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.

During somatic hypermutation (SHM), activation-induced deaminase (AID) mutates deoxycytidine on single-stranded DNA (ssDNA) generated by the transcription machinery, but the detailed mechanism remains unclear. Here we report a higher abundance of RNA polymerase II (Pol II) at the immunoglobulin heavy chain variable (Igh-V) region compared to the constant region and partially transcribed Igh RNAs, suggesting a slower Pol II progression at Igh-V that could result in some early/premature transcription termination after prolonged pausing/stalling of Pol II. Knocking down RNA exosome complexes, which could decrease premature transcription termination, leads to decreased SHM. Knocking down Spt5, which can augment premature transcription termination, leads to increases in both SHM and the abundance of ssDNA substrates. Collectively, our data support the model that, following the reduction of Pol II progression (pausing or stalling) at the Igh-V, additional steps such as premature transcription termination are involved in providing ssDNA substrates for AID during SHM.

The somatic hypermutation of Ig variable regions requires the activity of activation-induced cytidine deaminase (AID) which has previously been shown to preferentially deaminate WRC (W ‫؍‬ A/T, R ‫؍‬ A/G) motif hot spots in in vivo and in vitro assays. We compared mutation profiles of in vitro assays for the 3 flanking intron of VhJ558-Jh4 region to previously reported in vivo profiles for the same region in the Msh2 ؊/؊ Ung ؊/؊ mice that lack base excision and mismatch repair. We found that the in vitro and in vivo mutation profiles were highly correlated for the top (nontranscribed) strand, while for the bottom (transcribed) strand the correlation is far lower. We used an in silico model of AID activity to elucidate the relative importance of motif targeting in vivo. We found that the mutation process entails substantial complexity beyond motif targeting, a large part of which is captured in vitro. To elucidate the contribution of the sequence environment to the observed differences between the top and bottom strands, we analyzed intermutational distances. The bottom strand shows an approximately exponential distribution of distances in vivo and in vitro, as expected from a null model. However, the top strand deviates strongly from this distribution in that mutations approximately 50 nucleotides apart are greatly reduced, again both in vivo and in vitro, illustrating an important strand asymmetry. While we have confirmed that AID targeting of hot and cold spots is a key part of the mutation process, our results suggest that the sequence environment plays an equally important role.simulation ͉ somatic hypermutation ͉ variable-region

Health care personnel (HCP) are at increased risk for infection with SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), as a result of their exposure to patients or community contacts with COVID-19 (1,2). Since the first confirmed case of COVID-19 in Minnesota was reported on March 6, 2020, the Minnesota Department of Health (MDH) has required health care facilities* to report HCP † exposures to persons with confirmed COVID-19 for exposure risk assessment and to enroll HCP with higher-risk exposures into quarantine and symptom monitoring. During March 6-July 11, MDH and 1,217 partnering health care facilities assessed 21,406 HCP exposures; among these, 5,374 (25%) were classified as higher-risk § (3). Higher-risk exposures involved direct patient care (66%) and nonpatient care interactions (e.g., with coworkers and social and household contacts) (34%). Within 14 days following a higher-risk exposure, nearly one third (31%) of HCP who were enrolled in monitoring reported COVID-19-like symptoms, ¶ and more than one half (52%) of enrolled HCP with symptoms received positive * Health care facilities as defined by MDH include acute care hospitals, critical access hospitals, long-term acute care hospitals, skilled nursing facilities, assisted living facilities, group homes, adult foster care, treatment facilities, dialysis centers, outpatient clinics, dental clinics, home health care, and hospice. † HCP as defined by MDH include, but are not limited to, emergency medical service personnel, nurses, nursing assistants, physicians, technicians, therapists, phlebotomists, pharmacists, students and trainees, contractual staff members not employed by the health care facility, and persons not directly involved in patient care, but who could be exposed to infectious agents that can be transmitted in the health care setting (e.g., clerical, dietary, environmental services, laundry, security, engineering and facilities management, administrative, billing, and volunteer personnel). HCP does not include clinical laboratory personnel. § During February 8-May 18, 2020, CDC exposure risk assessment guidance included medium-and high-risk categories, with risk level based on PPE worn and type of potential contact with a person with confirmed COVID-19. On May 19, CDC's risk assessment was updated to include a single higher-risk exposure category to include close (within 6 feet), prolonged (≥15 minutes or of any duration during an aerosol-generating procedure) contact with a person with confirmed COVID