SUMMARY Interleukin 2 (IL-2) promotes Foxp3+-regulatory T (Treg) cell responses, but inhibits T follicular helper (TFH) cell development. However, it is not clear how IL-2 affects T follicular regulatory (TFR) cells, a cell type with properties of both Treg and TFH cells. Using an influenza infection model, we demonstrated that high IL-2 concentrations at the peak of the infection prevented TFR cell development by a Blimp-1–dependent mechanism. However, once the immune response resolved, some Treg cells down-regulated CD25, up-regulated Bcl-6 and differentiated into TFR cells, which then migrated into the B cell follicles to prevent the expansion of self-reactive B cell clones. Thus, unlike its effects on conventional Treg cells, IL-2 inhibits TFR cell responses.

Although B cells expressing the IFNγR or the IFNγ-inducible transcription factor T-bet promote autoimmunity in Systemic Lupus Erythematosus (SLE)-prone mouse models, the role for IFNγ signaling in human antibody responses is unknown. We show that elevated levels of IFNγ in SLE patients correlate with expansion of the T-bet expressing IgDnegCD27negCD11c+CXCR5neg (DN2) pre-antibody secreting cell (pre-ASC) subset. We demonstrate that naïve B cells form T-bethi pre-ASCs following stimulation with either Th1 cells or with IFNγ, IL-2, anti-Ig and TLR7/8 ligand and that IL-21 dependent ASC formation is significantly enhanced by IFNγ or IFNγ-producing T cells. IFNγ promotes ASC development by synergizing with IL-2 and TLR7/8 ligands to induce genome-wide epigenetic reprogramming of B cells, which results in increased chromatin accessibility surrounding IRF4 and BLIMP1 binding motifs and epigenetic remodeling of IL21R and PRDM1 loci. Finally, we show that IFNγ signals poise B cells to differentiate by increasing their responsiveness to IL-21.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

34Although B cells expressing the IFNgR or the IFNg-inducible transcription factor T-bet drive autoimmunity 35 in Systemic Lupus Erythematosus (SLE)-prone mouse models, the role for IFNg signaling in human 36 antibody responses is unknown. We show that elevated levels of IFNg in SLE patients correlate with 37 expansion of the T-bet expressing IgD neg CD27 neg CD11c + CXCR5 neg (DN2) pre-antibody secreting cell 38 (pre-ASC) subset. We demonstrate that naïve B cells form T-bet hi pre-ASCs following stimulation with 39 either Th1 cells or with IFNg, IL-2, anti-Ig and TLR7/8 ligand and that IL-21 dependent ASC formation is 40 significantly enhanced by IFNg or IFNg-producing T cells. IFNg promotes ASC development by 41 synergizing with IL-2 and TLR7/8 ligands to induce genome-wide epigenetic reprogramming of B cells, 42 which results in increased chromatin accessibility surrounding IRF4 and BLIMP1 binding motifs and 43 epigenetic remodeling of IL21R and PRDM1 loci. Finally, we show that IFNg signals poise B cells to 44 differentiate by increasing their responsiveness to 45 46 50 adaptive arms of the immune system, which ultimately leads to loss of immune tolerance in B and T 51 lymphocytes and the production of autoantibodies (Abs) by Ab-secreting B cells (ASCs) (1). The hallmark 52 SLE autoAbs recognize nuclear proteins and nucleic acids (2), which are also ligands for TLR7 and TLR9 53 that are expressed by innate immune cells and B cells (3). SLE autoAbs bound to their autoAgs form 54 immune complexes, which are responsible for many of the clinical manifestations of SLE, particularly 55 those associated with organ damage (2). Consistent with the important role for B cells and ASCs in SLE 56 pathogenesis (4), the only new drug approved to treat SLE in decades, Belimumab, targets B cells. 58Inflammatory cytokines and chemokines also contribute to SLE pathogenesis (5). SLE patient PBMCs 59 often exhibit a type I interferon (IFN) transcriptional signature and systemic IFNa is elevated in many 60 patients (6). It is less well appreciated that IFNg is also increased in some SLE patients (7-9) and that a 61 distinct IFNg transcription signature can be detected in PBMCs from a portion of SLE patients (10, 11). 62Interestingly, elevated serum IFNg can be observed years before IFNa or autoAbs are detected in SLE 63 patients and much earlier than clinical disease (12, 13). Consistent with these observations, B cells from 64 SLE patients can exhibit signs of prior IFNg exposure. For example, CXCR3 and T-bet, two IFNg-inducible 65 proteins (14), are more highly expressed by circulating B cells from SLE patients compared to healthy 66 controls (8,(15)(16)(17)(18)(19). Moreover, data from mouse SLE models show that clinical disease is dependent on 67 B cell-specific expression of the IFNgR and the IFNg-induced transcription factors and T-68 bet in some (23, 24) but not all (21, 25) models. Taken together, these data suggest that IFNg-driven 69 inflammation may contribute to SLE B cell-driven pathophysiology. 71Two populations of...

Coordinate regulation of PARPs-1/2 and PARG is required for cellular responses to genotoxic stress. While PARPs-1/2 are regulated by DNA breaks and covalent modifications, mechanisms of PARG regulation are poorly understood. We report here discovery of a PARG regulatory segment far removed linearly from residues involved in catalysis. Expression and analysis of human PARG segments identified a minimal catalytically active C-terminal PARG (hPARG59) containing a 16-residue N-terminal mitochondrial targeting sequence (MTS). Deletion analysis and site-directed mutagenesis revealed that the MTS, specifically hydrophobic residues L473 and L474 were required for PARG activity. This region of PARG was termed "Regulatory Segment/MTS" (REG/ MTS). The overall α-helical composition of hPARG59, determined by circular dichroism (CD), was unaffected by mutation of the REG/MTS leucine residues, suggesting that activity loss was not due to incorrect protein folding. REG/MTS was predicted to be in a loop conformation since the CD spectra of mutant Δ1-16 lacking the REG/MTS showed a higher α-helical content compared to hPARG59, indicating a secondary structure other than α-helix for this segment. Deletion of the REG/MTS from full-length hPARG111 also resulted in complete loss of activity, indicating that all PARG isoforms are subject to regulation at this site. Presence of the REG/MTS raises the possibility that PARG activity is regulated by interactions of PARPs-1/2 and other proteins at this site, raises interesting questions concerning mitochondrial PARG since MTS residues are often removed after transport, and offers a potentially novel site for drug targeting of PARG.The synthesis of polymers of ADP-ribose (PAR)1 is an immediate cellular response to DNA strand breaks caused by oxidative stress, ionizing radiation or alkylating agents (1-3). A primary enzyme involved in this post-translational modification is poly (ADP-ribose) polymerase-1 (PARP-1), which belongs to a growing family of PARPs that includes a second member (PARP-2) that also responds to DNA strand breaks (4,5). Upon binding to DNA strand breaks, PARPs-1/2 use nicotinamide adenine dinucleotide (NAD + ) as a substrate to synthesize PAR targeted to the PARPs themselves and other nuclear acceptor proteins, such as histones, p53 and enzymes involved in DNA repair (6). PARP-1 is inactivated and released from the DNA strand break by its automodification. Poly(ADP- † Supported by research grant CA 43894 from the National Institutes of Health to M.K.J.To whom correspondence should be addressed: Arizona Cancer Center Room 3985, University of Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724, USA. Tel: (520) 626-5957. Fax: (520) 626-8657. mjacobson@pharmacy.arizona.edu. 1 Abbreviations: ADP, adenosine-5′-diphosphate; PAR, poly(ADP-ribose); PARG, poly(ADP-ribose) glycohydrolase; hPARG, human PARG; PARP-1/2, poly(ADP-ribose) polymerase 1 and 2; ADPR, ADP-ribose; ADPRT, ADP-ribosyltransferase; NAD + , nicotinamide adenine dinucleotide; MTS, mitochondrial targeting...

In this study, we investigated the role of CD38 in a pristane-induced murine model of lupus. CD38-deficient (Cd38−/−) but not ART2-deficient (Art2−/−) mice developed less severe lupus compared to wild type (WT) mice, and their protective phenotype consisted of (i) decreased IFN-I-stimulated gene expression, (ii) decreased numbers of peritoneal CCR2hiLy6Chi inflammatory monocytes, TNF-α-producing Ly6G+ neutrophils and Ly6Clo monocytes/macrophages, (iii) decreased production of anti-single-stranded DNA and anti-nRNP autoantibodies, and (iv) ameliorated glomerulonephritis. Cd38−/− pristane-elicited peritoneal exudate cells had defective CCL2 and TNF-α secretion following TLR7 stimulation. However, Tnf-α and Cxcl12 gene expression in Cd38−/− bone marrow (BM) cells was intact, suggesting a CD38-independent TLR7/TNF-α/CXCL12 axis in the BM. Chemotactic responses of Cd38−/− Ly6Chi monocytes and Ly6G+ neutrophils were not impaired. However, Cd38−/− Ly6Chi monocytes and Ly6Clo monocytes/macrophages had defective apoptosis-mediated cell death. Importantly, mice lacking the cation channel TRPM2 (Trpm2−/−) exhibited very similar protection, with decreased numbers of PECs, and apoptotic Ly6Chi monocytes and Ly6Clo monocytes/macrophages compared to WT mice. These findings reveal a new role for CD38 in promoting aberrant inflammation and lupus-like autoimmunity via an apoptosis-driven mechanism. Furthermore, given the implications of CD38 in the activation of TRPM2, our data suggest that CD38 modulation of pristane-induced apoptosis is TRPM2-dependent.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.

Lung-derived dendritic cells carry influenza antigens to the spleen after egressing the lymph node by an S1P/S1PR-dependent mechanism.