The DNA Glycosylases Ogg1 and Nth1 Do Not Contribute to Ig Class Switching in Activated Mouse Splenic B Cells

During activation of B cells to undergo class switching, B cell metabolism is increased, and levels of reactive oxygen species (ROS) are increased. ROS can oxidize DNA bases resulting in substrates for the DNA glycosylases Ogg1 and Nth1. Ogg1 and Nth1 excise oxidized bases, and nick the resulting abasic sites, forming single-strand DNA breaks (SSBs) as intermediates during the repair process. In this study, we asked whether splenic B cells from mice deficient in these two enzymes would show altered class switching and decreased DNA breaks in comparison with wild-type mice. As the c-myc gene frequently recombines with the IgH S region in B cells induced to undergo class switching, we also analyzed the effect of deletion of these two glycosylases on DSBs in the c-myc gene. We did not detect a reduction in S region or c-myc DSBs or in class switching in splenic B cells from Ogg1- or Nth1-deficient mice or from mice deficient in both enzymes

In Vivo Binding and Retention of CD4-Specific DARPin 57.2 in Macaques

The recently described Designed Ankyrin Repeat Protein (DARPin) technology can produce highly selective ligands to a variety of biological targets at a low production cost.To investigate the in vivo use of DARPins for future application to novel anti-HIV strategies, we identified potent CD4-specific DARPins that recognize rhesus CD4 and followed the fate of intravenously injected CD4-specific DARPin 57.2 in rhesus macaques. The human CD4-specific DARPin 57.2 bound macaque CD4(+) cells and exhibited potent inhibitory activity against SIV infection in vitro. DARPin 57.2 or the control E3_5 DARPin was injected into rhesus macaques and the fate of cell-free and cell-bound CD4-specific DARPin was evaluated. DARPin-bound CD4(+) cells were detected in the peripheral blood as early as 30 minutes after the injection, decreasing within 6 hours and being almost undetectable within 24 hours. The amount of DARPin bound was dependent on the amount of DARPin injected. CD4-specific DARPin was also detected on CD4(+) cells in the lymph nodes within 30 minutes, which persisted with similar kinetics to blood. More extensive analysis using blood revealed that DARPin 57.2 bound to all CD4(+) cell types (T cells, monocytes, dendritic cells) in vivo and in vitro with the amount of binding directly proportional to the amount of CD4 on the cell surface. Cell-free DARPins were also detected in the plasma, but were rapidly cleared from circulation.We demonstrated that the CD4-specific DARPin can rapidly and selectively bind its target cells in vivo, warranting further studies on possible clinical use of the DARPin technology

Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells

Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma

DNA deaminases: AIDing hormones in immunity and cancer

It is well established that hormones can cause cancer, much less known is how they induce this change in our somatic cells. This review highlights the recent finding that estrogen can exert its DNA-damaging potential by directly activating DNA deaminases. This recently discovered class of proteins deaminate cytosine to uracil in DNA, and are essential enzymes in the immune system. The enhanced production of a given DNA deaminase, induced by estrogen, can lead not only to a more active immune response, but also to an increase in mutations and oncogenic translocations. Identifying the direct molecular link between estrogen and a mutation event provides us with new targets for studying and possibly inhibiting the pathological side-effects of estrogen

MSH2/MSH6 Complex Promotes Error-Free Repair of AID-Induced dU:G Mispairs as well as Error-Prone Hypermutation of A:T Sites

Mismatch repair of AID-generated dU:G mispairs is critical for class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The generation of a previously unavailable Msh2−/−Msh6−/− mouse has for the first time allowed us to examine the impact of the complete loss of MutSα on lymphomagenesis, CSR and SHM. The onset of T cell lymphomas and the survival of Msh2−/−Msh6−/− and Msh2−/−Msh6−/−Msh3−/− mice are indistinguishable from Msh2−/− mice, suggesting that MSH2 plays the critical role in protecting T cells from malignant transformation, presumably because it is essential for the formation of stable MutSα heterodimers that maintain genomic stability. The similar defects on switching in Msh2−/−, Msh2−/−Msh6−/− and Msh2−/−Msh6−/−Msh3−/− mice confirm that MutSα but not MutSβ plays an important role in CSR. Analysis of SHM in Msh2−/−Msh6−/− mice not only confirmed the error-prone role of MutSα in the generation of strand biased mutations at A:T bases, but also revealed an error-free role of MutSα when repairing some of the dU:G mispairs generated by AID on both DNA strands. We propose a model for the role of MutSα at the immunoglobulin locus where the local balance of error-free and error-prone repair has an impact in the spectrum of mutations introduced during Phase 2 of SHM

Antibody potency, effector function, and combinations in protection and therapy for SARS-CoV-2 infection in vivo

SARS-CoV-2, the causative agent of COVID-19, has been responsible for over 42 million infections and 1 million deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here, we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a Syrian hamster model of SARS-CoV-2 and in a mouse-adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). Antibody combinations were effective for prevention and in therapy when administered early. However, in vitro antibody neutralization potency did not uniformly correlate with in vivo protection, and some hu-mAbs were more protective in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors contributes to optimal protection against SARS-CoV-2 MA. The data indicate that intact effector function can affect hu-mAb protective activity and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention

Jak3 Is Involved in Dendritic Cell Maturation and CCR7-Dependent Migration

BACKGROUND: CCR7-mediated signalling is important for dendritic cell maturation and homing to the lymph nodes. We have previously demonstrated that Jak3 participates in the signalling pathway of CCR7 in T lymphocytes. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we used Jak3(-/-) mice to analyze the role of Jak3 in CCR7-mediated dendritic cells migration and function. First, we found no differences in the generation of DCs from Jak3(-/-) bone marrow progenitors, when compared to wild type cells. However, phenotypic analysis of the bone marrow derived DCs obtained from Jak3(-/-) mice showed reduced expression of co-stimulatory molecules compared to wild type (Jak3(+/+)). In addition, when we analyzed the migration of Jak3(-/-) and Jak3(+/+) mature DCs in response to CCL19 and CCL21 chemokines, we found that the absence of Jak3 results in impaired chemotactic responses both in vitro and in vivo. Moreover, lymphocyte proliferation and contact hypersensitivity experiments showed that DC-mediated T lymphocyte activation is reduced in the absence of Jak3. CONCLUSION/SIGNIFICANCE: Altogether, our data provide strong evidence that Jak3 is important for DC maturation, migration and function, through a CCR7-mediated signalling pathway

HSV-2 Infection of Dendritic Cells Amplifies a Highly Susceptible HIV-1 Cell Target

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α4β7 on lymphocytes. α4β7 can be engaged by HIV-1 on the cell-surface and CD4+ T cells expressing high levels of this integrin (α4β7high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α4β7high CD4+ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c+ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α4β7 expression on CD4+ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α4β7highCD4+ T cells. These factors may play a role in increasing the susceptibility to HIV-1