Germinal Center Research Articles

Vaccines combining slow delivery and follicle targeting of antigens increase germinal center B cell clonal diversity and clonal expansion.

Vaccines incorporating slow delivery, multivalent antigen display, or immunomodulation through adjuvants have an important role to play in shaping the humoral immune response. Here we analyzed mechanisms of action of a clinically relevant combination adjuvant strategy, where phosphoserine (pSer)-tagged immunogens bound to aluminum hydroxide (alum) adjuvant (promoting prolonged antigen delivery to draining lymph nodes) are combined with a potent saponin nanoparticle adjuvant termed SMNP (which alters lymph flow and antigen entry into lymph nodes). When employed with a stabilized HIV Env trimer antigen in mice, this combined adjuvant approach promoted substantial enhancements in germinal center (GC) and antibody responses relative to either adjuvant alone. Using scRNA-seq and scBCR-seq, we found that the alum-pSer/SMNP combination both increased the diversity of GC B cell clones and increased GC B cell clonal expansion, coincident with increases in the expression of Myc and the proportion of S-phase GC B cells. To gain insight into the source of these changes in the GC response, we analyzed antigen biodistribution and structural integrity in draining lymph nodes and found that the combination adjuvant approach, but not alum-pSer delivery or SMNP alone, promoted accumulation of highly intact antigen on follicular dendritic cells, reflecting an integration of the slow antigen delivery and altered lymph node uptake effects of these two adjuvants. These results demonstrate how adjuvants with complementary mechanisms of action impacting vaccine biodistribution and kinetics can synergize to enhance humoral immunity.

Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient germinal center B cell responses.

The mammalian Brg1/Brm-associated factor (BAF) complexes are major regulators of nucleosomal remodeling that are commonly mutated in several cancers, including germinal center (GC)-derived B cell lymphomas. However, the specific roles of different BAF complexes in GC B cell biology are not well understood. Here we show that the AT-rich interaction domain 1a (Arid1a) containing canonical BAF (cBAF) complex is required for maintenance of GCs and high-affinity antibody responses. While Arid1a-deficient B cells undergo initial activation, they fail to sustain the GC program. Arid1a establishes permissive chromatin landscapes for B cell activation and is concomitantly required to suppress inflammatory gene programs. The inflammatory signatures instigated by Arid1a deficiency promoted the recruitment of neutrophils and inflammatory monocytes. Dampening of inflammatory cues through interleukin-1β blockade or glucocorticoid receptor agonist partially rescued Arid1a-deficient GCs, highlighting a critical role for inflammation in impeding GCs. Our work reveals essential functions of Arid1a-dependent cBAF in promoting efficient GC responses.

TFEB activation hallmarks antigenic experience of B lymphocytes and directs germinal center fate decisions

Ligation of the B cell antigen receptor (BCR) initiates humoral immunity. However, BCR signaling without appropriate co-stimulation commits B cells to death rather than to differentiation into immune effector cells. How BCR activation depletes potentially autoreactive B cells while simultaneously primes for receiving rescue and differentiation signals from cognate T lymphocytes remains unknown. Here, we use a mass spectrometry-based proteomic approach to identify cytosolic/nuclear shuttling elements and uncover transcription factor EB (TFEB) as a central BCR-controlled rheostat that drives activation-induced apoptosis, and concurrently promotes the reception of co-stimulatory rescue signals by supporting B cell migration and antigen presentation. CD40 co-stimulation prevents TFEB-driven cell death, while enhancing and prolonging TFEB’s nuclear residency, which hallmarks antigenic experience also of memory B cells. In mice, TFEB shapes the transcriptional landscape of germinal center B cells. Within the germinal center, TFEB facilitates the dark zone entry of light-zone-residing centrocytes through regulation of chemokine receptors and, by balancing the expression of Bcl-2/BH3-only family members, integrates antigen-induced apoptosis with T cell-provided CD40 survival signals. Thus, TFEB reprograms antigen-primed germinal center B cells for cell fate decisions.

Spatial technologies to evaluate the HIV-1 reservoir and its microenvironment in the lymph node.

The presence of the HIV-1 reservoir, a group of immune cells that contain intact, integrated, and replication-competent proviruses, is a major challenge to cure HIV-1. HIV-1 reservoir cells are largely unaffected by the cytopathic effects of viruses, antiviral immune responses, or antiretroviral therapy (ART). The HIV-1 reservoir is seeded early during HIV-1 infection and augmented during active viral replication. CD4+ T cells are the primary target for HIV-1 infection, and recent studies suggest that memory T follicular helper cells within the lymph node, more precisely in the B cell follicle, harbor integrated provirus, which contribute to viral rebound upon ART discontinuation. The B cell follicle, more specifically the germinal center, possesses a unique environment because of its distinct property of being partly immune privileged, potentially allowing HIV-1-infected cells within the lymph nodes to be protected from CD8+ T cells. This modified immune response in the germinal center of the follicle is potentially explained by the exclusion of CD8+ T cells and the presence of T regulatory cells at the junction of the follicle and extrafollicular region. The proviral makeup of HIV-1-infected cells is similar in lymph nodes and blood, suggesting trafficking between these compartments. Little is known about the cell-to-cell interactions, microenvironment of HIV-1-infected cells in the follicle, and trafficking between the lymph node follicle and other body compartments. Applying a spatiotemporal approach that integrates genomics, transcriptomics, and proteomics to investigate the HIV-1 reservoir and its neighboring cells in the lymph node has promising potential for informing HIV-1 cure efforts.

Sex-based difference in immune responses and efficacy of the pneumococcal conjugate vaccine.

Vaccine-mediated protection and susceptibility to Streptococcus pneumoniae (pneumococcus) infections are influenced by biological sex. The incidence of invasive pneumococcal disease remains higher in males compared to females even after the introduction of the pneumococcal conjugate vaccine (PCV). However, sex-based differences in the immune response to this conjugate vaccine remain unexplored. To investigate those differences, we vaccinated adult male and female mice with PCV and assessed cellular and humoral immune responses. Compared to females, male mice displayed lower levels of T follicular helper cells, germinal center B cells and plasmablasts, which are all required for antibody production following vaccination. This was linked to lower IgG and IgM levels against pneumococci and lower isotype switching to IgG3 in vaccinated males. Due to lower antibody levels, sera of vaccinated male mice had lower efficacy in several anti-pneumococcal functions including neutralization of bacterial binding to pulmonary epithelial cells as well as direct cytotoxicity against S. pneumoniae. Importantly, while the vaccine was highly protective in females, vaccinated males succumbed to infection more readily and were more susceptible to both lung-localized infection and systemic spread following S. pneumoniae challenge. These findings identify sex-based differences in immune responses to PCV that can inform future vaccine strategies.

A nucleoside-modified mRNA vaccine forming rabies virus-like particle elicits strong cellular and humoral immune responses against rabies virus infection in mice

ABSTRACT Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.

Genetic modification of primary human B cells to model the process of B cell development in germinal centers

The main stages of maturation of antigen-specific B cells occur in the germinal centers of the lymph nodes. During the process of differentiation, a decision is made on which path the B cells will take to develop further. They will either turn into short-lived plasmablasts or memory B cells or plasma cells. The relationship between these processes is very important for the development of a productive humoral immune response. The goal of the work was to create a system that is capable of simulating ex vivo processes occurring in germinal centers. We used primary B cells from human peripheral blood as starting material. B lymphocytes were stimulated in vitro using feeder cells carrying CD40L molecules and recombinant IL-21. Upon IL-21/CD40L stimulation, B lymphocytes changed their morphology, surface phenotype, and functional activity. After active expansion for 10 days, further cell growth stopped, and after some time they died. To generate stably proliferating B cells, we used lentiviral transduction of IL-21/CD40L stimulated IgM+ B cells. For this purpose, lentivirus preparations were obtained that carried a cassette consisting of the BCL6 and BCL2L1 genes, separated by a sequence encoding the self-cutting peptide P2A, as well as a GFP reporter gene separated from the target genes by an IRES element. The cassette used ensured the synthesis of the Bcl-6 transcription factor and the Bcl-XL protein in target cells. The Bcl-6 repressor prevented B cells from undergoing terminal differentiation and becoming plasma cells, and the Bcl-XL protein had an anti-apoptotic effect. Transduced B cells proliferated for more than a month and maintained a plasmablast phenotype. Forty-two days after the start of stimulation, transduced B cells remained GFP-positive, coexpressed CD27 and CD38 antigens, carried surface CD20 and IgM, intracellular Bcl-6, Bcl-XL and IgM, retained IgM secretion, but remained negative for surface and intracellular IgG. The proven stimulation system will allow us to simulate key aspects of B cell development in germinal centers to study the formation of B cell memory, which will ultimately facilitate the development of effective vaccines.

Sphingosine-1-Phosphate Receptor 4 links neutrophils and early local inflammation to lymphocyte recruitment into the draining lymph node to facilitate robust germinal center formation.

The successful development of germinal centers (GC) relies heavily on innate mechanisms to amplify the initial inflammatory cascade. In addition to their role in antigen presentation, innate cells are essential for the redirection of circulating lymphocytes toward the draining lymph node (dLN) to maximize antigen surveillance. Sphingosine-1-Phosphate (S1P) and its receptors (S1PR1-5) affect various aspects of immunity; however, the role of S1PR4 in regulating an immune response is not well understood. Here we use a footpad model of localized TH1 inflammation to carefully monitor changes in leukocyte populations within the blood, the immunized tissue, and the dLN. Within hours of immunization, neutrophils failed to adequately mobilize and infiltrate into the footpad tissue of S1PR4-/- mice, thereby diminishing the local vascular changes thought to be necessary for redirecting circulating cells toward the inflamed region. Neutrophil depletion with anti-Ly6G antibodies significantly reduced early tissue edema as well as the redirection and initial accumulation of naïve lymphocytes in dLN of WT mice, while the effects were less prominent or absent in S1PR4-/- dLN. Adoptive transfer experiments further demonstrated that the lymphocyte homing deficiencies in vivo were not intrinsic to the donor S1PR4-/- lymphocytes, but were instead attributed to differences within the S1PR4-deficient host. Reduced cell recruitment in S1PR4-/- mice would seed the dLN with fewer antigen-respondent lymphocytes and indeed, dLN hypertrophy at the peak of the immune response was severely diminished, with attenuated GC and activation pathways in these mice. Histological examination of the S1PR4-/- dLN also revealed an underdeveloped vascular network with reduced expression of the leukocyte tethering ligand, PNAd, within high endothelial venule regions, suggesting inadequate growth of the dLN meant to support a robust GC response. Thus, our study reveals that S1PR4 may link early immune modulation by neutrophils to the initial recruitment of circulating lymphocytes and downstream expansion and maturation of the dLN, thereby contributing to optimal GC development during an adaptive response.

Follicular Immune Landscaping Reveals a Distinct Profile of FOXP3hiCD4hi T Cells in Treated Compared to Untreated HIV.

Follicular helper CD4hi T cells (TFH) are a major cellular pool for the maintenance of the HIV reservoir. Therefore, the delineation of the follicular (F)/germinal center (GC) immune landscape will significantly advance our understanding of HIV pathogenesis. We have applied multiplex confocal imaging, in combination with the relevant computational tools, to investigate F/GC in situ immune dynamics in viremic (vir-HIV), antiretroviral-treated (cART HIV) People Living With HIV (PLWH) and compare them to reactive, non-infected controls. Lymph nodes (LNs) from viremic and cART PLWH could be further grouped based on their TFH cell densities in high-TFH and low-TFH subgroups. These subgroups were also characterized by different in situ distributions of PD1hi TFH cells. Furthermore, a significant accumulation of follicular FOXP3hiCD4hi T cells, which were characterized by a low scattering in situ distribution profile and strongly correlated with the cell density of CD8hi T cells, was found in the cART-HIV low-TFH group. An inverse correlation between plasma viral load and LN GrzBhiCD8hi T and CD16hiCD15lo cells was found. Our data reveal the complex GC immune landscaping in HIV infection and suggest that follicular FOXP3hiCD4hi T cells could be negative regulators of TFH cell prevalence in cART-HIV.

Cedrol in ginger (Zingiber officinale) as a promising hair growth drug: The effects of oral and external administration on hair regeneration and its mechanism

Ginger is an important cooking spice and herb worldwide, and scientific research has gradually confirmed the effect of ginger on preventing hair loss. Cedrol (CE) is a small sesquiterpene molecule in ginger and its external administration (EA) has shown hope in promoting hair growth, and alternative administration mode has become a potential treatment scheme to improve the efficacy of CE. The purpose of this study is to evaluate the effects of oral administration (OA) and EA of CE on hair regeneration of C57BL/6 alopecia areata (AA) mice induced by cyclophosphamide (CP) and to clarify the potential hair growth mechanism of CE in AA model in vitro and in vivo. The results showed that CE-OA has a shorter hair-turning black time and faster hair growth rate, and can lessen hair follicle damage induced by CP and promote hair follicle cell proliferation. Its effect is superior to CE-EA. At the same time, CE can increase the cytokines IFN-γ, IL-2, and IL-7 in the serum of mice, and decrease the expression of adhesion factors ICAM-1 and ELAM-1, thus alleviating the immunosuppression induced by CP. Mechanism research shows that CE regulates the JAK3/STAT3 signaling pathway, activates the Wnt3α/β-catenin germinal center, and ameliorates oxidative stress induced by CP, thus promoting the proliferation of hair follicle cells and reversing AA. These results provide a theoretical basis for understanding the anti-AA mechanism of CE-OA, indicating that CE can be used as raw material for developing oral hair growth drugs.

Bruton's tyrosine kinase inhibitor zanubrutinib-based regimens in relapsed/refractory primary diffuse large B-cell lymphoma of central nervous system.

30 Background: Patients with relapsed/refractory primary central nervous system lymphoma (R/R PCNSL) usually have a poor prognosis. Ibrutinib-based regimens revealed promising efficacy but showed off-target effects and drug resistance in some cases. Here, we conducted a retrospective study to evaluate the efficacy and safety of regimens based on second-generation bruton's tyrosine kinase (BTK) inhibitor zanubrutinib in R/R PCNSL. Methods: We respectively reviewed 23 patients with R/R PCNSL treated with zanubrutinib in our center from Apr. 2021 to Jan. 2024. The primary end points were overall response rate (ORR) and progression-free survival (PFS). Secondary end points included complete response rate (CRR), disease control rate (DCR), overall survival (OS) and adverse events (AEs). Response was assessed according to International Primary CNS Lymphoma Collaborative Group criteria. The severity of AEs was graded using the NCI Common Toxicity Criteria, version 5.0. Validated next-generation sequencing (NGS) panels were used to analyze the mutational status of the BCR pathway genes or other solid tumor-related genes. Results: The median follow-up at the cutoff date (15 Jan 2024) was 17.0 months (range, 2-33 months). The ORR, CRR and DCR were 78.3%, 39.1% and 91.3%. The median PFS was 31.0 months, but the median OS was not reached. Compared to germinal center B-cell like (GCB) group, non-GCB group showed a better ORR (76.9% vs 66.7%) and CRR (46.2% VS 33.3%). Multivariate analysis revealed that overall response (vs. no response, HR=0.20, P=0.027), long duration of zanubrutinib (≥6months vs. 2-5 months, HR=0.07, P=0.009) and refractory disease status (vs. relapse disease status, HR=0.12, P=0.014) were independent factors for longer PFS. The most commonly mutated genes are MYD88(15/17,88.2%), PIM1(9/17 52.9%), CD79B (8/17,47.1%), ETV6(7/17,41.2%), BTG1(5/17,29.4%) and GNA13(5/17,29.41%). The mutational status of MYD88/CD79B did not lead to any difference in PFS and OS. We obtained the Tumor Mutational Burden (TMB) information from 11 patients. The median TMB was 15.96 (range: 3.82-28.11) muts/Mb. Kaplan-Meier analysis demonstrated that PFS was significantly longer in patients with higher TMB (≥15.96 muts/Mb) after zanubrutinib-based treatment(P=0.005). Grade ≥3 and serious treatment-emergent AEs (TEAEs) were not found. The most commonly TEAEs were hematologic toxicity (8/23,34.8%) and skin rash (5/23,21.7%). Conclusions: Our study demonstrates that zanubrutinib-based therapy is effective and safe for the treatment of R/R PCNSL. We advocate for the prolonged administration of zanubrutinib to maintain treatment response. Patients with higher TMB derive greater benefits from zanubrutinib-based regimens.

B cell senescence promotes age-related changes in oral microbiota.

In recent years, there has been increasing attention towards understanding the relationship between age-related alterations in the oral microbiota and age-associated diseases, with reports emphasizing the significance of maintaining a balanced oral microbiota for host health. However, the precise mechanisms underlying age-related changes in the oral microbiota remain elusive. We recently reported that cellular senescence of ileal germinal center (GC) B cells, triggered by the persistent presence of commensal bacteria, results in diminished IgA production with aging and subsequent alterations in the gut microbiota. Consequently, we hypothesize that a similar phenomenon may occur in the oral cavity, potentially contributing to age-related changes in the oral microbiota. Examination of p16-luc mice, wherein the expression of the senescent cell marker p16INK4a can be visualized, raised under specific pathogen-free (SPF) or germ-free (GF) conditions, indicated that, unlike ileal GC B cells, the accumulation of senescent cells in GC B cells of cervical lymph nodes increases with age regardless of the presence of commensal bacteria. Furthermore, longitudinal studies utilizing the same individual mice throughout their lifespan revealed concurrent age-related alterations in the composition of the oral microbiota and a decline in salivary IgA secretion. Further investigation involving Rag1-/- mice transplanted with B cells from wild-type or p16INK4a and p21Waf1/Cip1 -double knockout mice unveiled that B cell senescence leads to reduced IgA secretion and alteration of the oral microbiota. These findings advance our understanding of the mechanism of age-associated changes in the oral microbiota and open up possibilities of their control.

B cells require DOCK8 to elicit and integrate T cell help when antigen is limiting.

Dedicator of cytokinesis 8 (DOCK8) immunodeficiency syndrome is characterized by a failure of the germinal center response, a process involving the proliferation and positive selection of antigen-specific B cells. Here, we describe how DOCK8-deficient B cells are blocked at a light-zone checkpoint in the germinal centers of immunized mice, where they are unable to respond to T cell-dependent survival and selection signals and consequently differentiate into plasma cells or memory B cells. Although DOCK8-deficient B cells can acquire and present antigen to initiate activation of cognate T cells, integrin up-regulation, B cell-T cell conjugate formation, and costimulation are insufficient for sustained B cell and T cell activation when antigen availability is limited. Our findings provide an explanation for the failure of the humoral response in DOCK8 immunodeficiency syndrome and insight into how the level of available antigen modulates B cell-T cell cross-talk to fine-tune humoral immune responses and immunological memory.

IL-17B alleviates the pathogenesis of systemic lupus erythematosus by inhibiting FASN-mediated differentiation of B cells.

The interleukin 17 (IL-17) family of cytokines has emerged as a critical player in autoimmune disease, including systemic lupus erythematosus (SLE). However, the role of IL-17B, a poorly understood cytokine, in the pathogenesis of SLE is still not known. In this study, we investigated the role of IL-17B in the activation and differentiation of B cells, and the pathogenesis of SLE. Intriguingly, IL-17B deficiency aggravated disease in lupus-prone mice and promoted the activation of B cells and the differentiation of germinal center B cells and plasma cells, while recombinant mouse IL-17B (rmIL-17B) significantly alleviated disease in lupus-prone mice. Mechanistically, rmIL-17B inhibited the activation of the Toll-like receptor and interferon pathways in B cells by downregulating fatty acid synthase-mediated (FASN-mediated) lipid metabolism. Loss of FASN significantly alleviated the disease in lupus-prone mice and inhibited the activation and differentiation of B cells. In addition, B cells had greater FASN expression and lower IL-17RB levels in patients with SLE than in healthy controls. Our study describes the role of IL-17B in regulating B cell activation and differentiation, and alleviating the onset of SLE. These findings will lay a theoretical foundation for further understanding of the pathogenesis of SLE.

Clinical characteristics and outcome of early-stage diffuse large B cell lymphoma of female genital track: A retrospective study of the Hellenic cooperative lymphoma group.

Involvement of female genital track (FGT) by diffuse large B cell lymphoma (DLBCL) represents an extremely rare diagnosis. Especially data regarding early-stage disease (i.e., IE, IIE) is very limited. Importantly, previous studies showed controversial results about the risk of central nervous system (CNS) relapse in this entity. Herein, we describe one of the largest reported real-world series of patients with early-stage FGT DLBCL aiming to investigate the clinicopathological characteristics, response to therapy and survival outcomes in the era of immunochemotherapy. We analyzed 21 consecutive patients with biopsy proven DLBCL from uterus or ovary classified as stage IE or IIE out of 1905 newly diagnosed DLBCL patients (1.1%). Uterine and ovarian localization was observed in 14 and seven patients, respectively. Median age was 66years (range 33-96); 9/21 (43%) were <55years. Regarding Cell of Origin DLBCL subtype, Germinal Center B-cell subtype was found in seven patients, non-GCB in 10 and non-classified in 4 patients. Median follow-up was 57months and 5-year overall survival, lymphoma specific survival and Freedom from Progression were 78%, 89% and 90%, respectively. There was no correlation of patients' characteristics with survival parameters. Interestingly, none of the patients experienced CNS relapse. Our results indicate that localized FGT DLBCL exhibits a good prognosis and may not increase the risk for secondary CNS involvement.

CX-5461 ameliorates disease in lupus-prone mice by triggering B-cell ferroptosis via p53-SLC7A11-ALOX12 pathway

CX-5461, a first-in-class compound, is widely recognized as a selective inhibitor of RNA polymerase I. Recently, it has been reported to possess novel immunosuppressive properties with significant therapeutic effects in transplantation immune rejection. However, the potential use of CX-5461 for Systemic Lupus Erythematosus (SLE) treatment remains unknown. In this study, we elucidated the mechanism underlying the therapeutic efficacy of CX-5461 in lupus. Our findings demonstrated that CX-5461 selectively targets B cells and effectively reduces the proportions of B cells, germinal center B cells, and plasma cells in MRL/MPJ-Faslpr and Resiquimod (R848)-induced lupus mice. Molecular studies revealed that CX-5461 modulates CD36-Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4)-mediated glycerolipid metabolism in B cells, triggering ferroptosis through the p53- Solute Carrier Family 7 Member 11 (SLC7A11)- Arachidonate 12-Lipoxygenase (ALOX12) pathway, thereby decreasing IgG and Anti-Double-Stranded Deoxyribonucleic Acid (dsDNA) antibody levels and attenuating lupus. Collectively, these results suggest that CX-5461 holds promise as an effective candidate for targeted therapy against lupus.

Repurposing NAMPT Inhibitors for Germinal Center B Cell-Like Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) includes the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, which differ in cell of origin, genetics, and clinical response. By screening the subtype-specific activity of 211 drugs approved or in active clinical development for other diseases, we identified inhibitors of nicotinamide phosphoribosyl transferase (NAMPTi) as active in a subset of GCB-DLBCL in vitro and in vivo. We validated three chemically distinct NAMPTis for their on-target activity based on biochemical and genetic rescue approaches and found the ratio between NAMPT and PARP1 RNA levels was predictive of NAMPTi sensitivity across DLBCL subtypes. Notably, the NAMPT:PARP1 transcript ratio predicts higher antitumor activity in BCL2-translocated GCB-DLBCL. Accordingly, pharmacologic and genetic inhibition of BCL2 was potently synergistic with NAMPT blockade. These data support the inhibition of NAMPT as a therapeutically relevant strategy for BCL2-translocated DLBCLs. Significance: Targeted therapies have emerged for the ABC subtype of DLBCL, but not for the GCB subtype, despite the evidence of a significant subset of high-risk cases. We identify a drug that specifically targets a subset of GCB-DLBCL and provide preclinical evidence for BCL2 translocations as biomarkers for their identification.

Orally dissolving film as a potential vaccine delivery carrier to prevent influenza virus infection

Orally dissolving films (ODF) are designed to be dissolved on the tongue and absorbed in the mouth. It offers multiple advantages over the commonly used needle-based vaccines, especially in terms of convenience allowing safe, painless, and easy self-administration. As the efficacy of ODF-encapsulated influenza vaccines has not been demonstrated, we assessed the protection elicited by inactivated influenza virus (A/PR/8/34, H1N1) vaccine delivered using ODFs in mice. Trehalose and pullulan components of the ODF ensured that the HA antigens of the inactivated PR8 virus retained their stability while ensuring the rapid release of the vaccines upon exposure to murine saliva. Mice were immunized thrice by placing the PR8-ODF on the tongues of mice at 4-week intervals, and vaccine-induced protection was evaluated upon lethal homologous challenge infection. The PR8-ODF vaccination elicited virus-specific serum IgG and IgA antibody responses, hemagglutinin inhibition (HAI), and viral neutralization. Upon challenge infection, ODF vaccination showed higher levels of IgG and IgA antibody responses in the lungs and antibody-secreting cell (ASC) responses in both lung and spleen compared to unimmunized controls. These results corresponded with the enhanced T cell and germinal center B cell responses in the lungs and spleens. Importantly, ODF vaccination significantly reduced lung virus titers and inflammatory cytokines (IFN-γ, IL-6) production compared to unvaccinated control. ODF vaccination ensured 100% survival and prevented weight loss in mice. These findings suggest that influenza vaccine delivery through ODFs could be a promising approach for oral vaccine development.

Targeting mechanistic target of rapamycin complex 2 attenuates immunopathology in Systemic Lupus Erythematosus.

We aim to explore the role of mechanistic target of rapamycin complex (mTORC) 2 in systemic lupus erythematosus (SLE) development, the in vivo regulation of mTORC2 by type I interferon (IFN) signaling in autoimmunity, and to use mTORC2 targeting therapy to ameliorate lupus-like symptoms in an in vivo lupus mouse model and an in vitro coculture model using human PBMCs. We first induced lupus-like disease in T cell specific Rictor, a key component of mTORC2, deficient mice by topical application of imiquimod (IMQ) and monitored disease development. Next, we investigated the changes of mTORC2 signaling and immunological phenotypes in type I IFNAR deficient Lpr mice. We then tested the beneficial effects of anti-Rictor antisense oligonucleotide (Rictor-ASO) in a mouse model of lupus: MRL/lpr mice. Finally, we examined the beneficial effects of RICTOR-ASO on SLE patients' PBMCs using an in vitro T-B cell coculture assay. T cell specific Rictor deficient mice have reduced age-associated B cells, plasma cells and germinal center B cells, and less autoantibody production than WT mice following IMQ treatment. IFNAR1 deficient Lpr mice have reduced mTORC2 activity in CD4+ T cells accompanied by restored CD4+ T cell glucose metabolism, partially recovered T cell trafficking, and reduced systemic inflammation. In vivo Rictor-ASO treatment improves renal function and pathology in MRL/lpr mice, along with improved immunopathology. In human SLE (N = 5) PBMCs derived T-B coculture assay, RICTOR-ASO significantly reduce immunoglobulin and autoantibodies production (P < 0.05). Targeting mTORC2 could be a promising therapeutic for SLE.

Control of germinal center B cell survival and IgE production by an adaptor molecule containing PH and SH2 domains, Aps/Sh2b2

The germinal centers (GCs) are structure found within secondary lymphoid organs and are important for the antibody-producing response against foreign antigens. In GCs, antigen-specific B cells proliferate intensely, inducing immunoglobulin class switching. Recent studies have shown that GCs are also an important site for class switching to IgE, which is implicated in allergy. However, the mechanisms by which IgE production is regulated in GCs remain unclear. Here, we found impairment in IgE-specific production and a reduction of GC B cells after immunization in mice deficient in the Aps/Sh2b2 gene encoding the Lnk/Sh2b family adaptor protein Aps. GC B cells express higher levels of the Aps gene than non-GC B cells, and cell death of Aps-/- GC B cells is enhanced compared to wild-type GC B cells. An in vitro culture system with purified Aps-/- B cells induced the same level of IgE production and frequencies of IgE+ B cells as wild-type B cells. We found that Aps deficiency in B cells resulted in augmented depletion of IgE+ blasts by B cell receptor crosslinking with anti-CD79b antibodies compared to wild-type IgE+ cells. These results suggest that Aps regulates IgE production by controlling the survival of GC B cells and IgE+ plasma cells and may serve as a potential therapeutic target to control IgE production.