Assembly and activation of EBV latent membrane protein 1.

Latent membrane protein 1 (LMP1) is the primary oncoprotein of Epstein-Barr virus (EBV) and plays versatile roles in the EBV life cycle and pathogenesis. Despite decades of extensive research, the molecular basis for LMP1 folding, assembly, and activation remains unclear. Here, we report cryo-electron microscopy structures of LMP1 in two unexpected assemblies: a symmetric homodimer and a higher-order filamentous oligomer. LMP1 adopts a non-canonical and unpredicted fold that supports the formation of a stable homodimer through tight and antiparallel intermolecular packing. LMP1 dimers further assemble side-by-side into higher-order filamentous oligomers, thereby allowing the accumulation and specific organization of the flexible cytoplasmic tails for efficient recruitment of downstream factors. Super-resolution microscopy and cellular functional assays demonstrate that mutations at both dimeric and oligomeric interfaces disrupt LMP1 higher-order assembly and block multiple LMP1-mediated signaling pathways. Our research provides a framework for understanding the mechanism of LMP1 and for developing potential therapies targeting EBV-associated diseases.

A neutrophil response linked to tumor control in immunotherapy.

Neutrophils accumulate in solid tumors, and their abundance correlates with poor prognosis. Neutrophils are not homogeneous, however, and could play different roles in cancer therapy. Here, we investigate the role of neutrophils in immunotherapy, leading to tumor control. We show that successful therapies acutely expanded tumor neutrophil numbers. This expansion could be attributed to a Sellhi state rather than to other neutrophils that accelerate tumor progression. Therapy-elicited neutrophils acquired an interferon gene signature, also seen in human patients, and appeared essential for successful therapy, as loss of the interferon-responsive transcription factor IRF1 in neutrophils led to failure of immunotherapy. The neutrophil response depended on key components of anti-tumor immunity, including BATF3-dependent DCs, IL-12, and IFNγ. In addition, we found that a therapy-elicited systemic neutrophil response positively correlated with disease outcome in lung cancer patients. Thus, we establish a crucial role of a neutrophil state in mediating effective cancer therapy.

Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

The pre-exposure SARS-CoV-2-specific T cell repertoire determines the quality of the immune response to vaccination.

SARS-CoV-2 infection and vaccination generates enormous host-response heterogeneity and an age-dependent loss of immune-response quality. How the pre-exposure T cell repertoire contributes to this heterogeneity is poorly understood. We combined analysis of SARS-CoV-2-specific CD4+ T cells pre- and post-vaccination with longitudinal T cell receptor tracking. We identified strong pre-exposure T cell variability that correlated with subsequent immune-response quality and age. High-quality responses, defined by strong expansion of high-avidity spike-specific T cells, high interleukin-21 production, and specific immunoglobulin G, depended on an intact naive repertoire and exclusion of pre-existing memory T cells. In the elderly, T cell expansion from both compartments was severely compromised. Our results reveal that an intrinsic defect of the CD4+ T cell repertoire causes the age-dependent decline of immune-response quality against SARS-CoV-2 and highlight the need for alternative strategies to induce high-quality T cell responses against newly arising pathogens in the elderly.

Regulatory T Cell Specificity Directs Tolerance versus Allergy against Aeroantigens in Humans.

FOXP3+ regulatory T cells (Tregs) maintain tolerance against self-antigens and innocuous environmental antigens. However, it is still unknown whether Treg-mediated tolerance is antigen specific and how Treg specificity contributes to the selective loss of tolerance, as observed in human immunopathologies such as allergies. Here, we used antigen-reactive T cell enrichment to identify antigen-specific human Tregs. We demonstrate dominant Treg-mediated tolerance against particulate aeroallergens, such as pollen, house dust mites, and fungal spores. Surprisingly, we found no evidence of functional impairment of Treg responses in allergic donors. Rather, major allergenic proteins, known to rapidly dissociate from inhaled allergenic particles, have a generally reduced capability to generate Treg responses. Most strikingly, in individual allergic donors, Th2 cells and Tregs always target disparate proteins. Thus, our data highlight the importance of Treg antigen-specificity for tolerance in humans and identify antigen-specific escape from Treg control as an important mechanism enabling antigen-specific loss of tolerance in human allergy.

Mutations in the transcription factor FOXO1 mimic positive selection signals to promote germinal center B cell expansion and lymphomagenesis.

The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas.

BCL6 controls contact-dependent help delivery during follicular T-B cell interactions.

BCL6 is required for development of follicular T helper (Tfh) cells to support germinal center (GC) formation. However, it is not clear what unique functions programmed by BCL6 can explain its absolute essentiality in T cells for GC formation. We found that ablation of one Bcl6 allele did not appreciably alter early T cell activation and follicular localization but inhibited GC formation and Tfh cell maintenance. BCL6 impinged on Tfh calcium signaling and also controlled Tfh entanglement with and CD40L delivery to B cells. Amounts of BCL6 protein and nominal frequencies of Tfh cells markedly changed within hours after strengths of T-B cell interactions were altered in vivo, while CD40L overexpression rectified both defective GC formation and Tfh cell maintenance because of the BCL6 haploinsufficiency. Our results reveal BCL6 functions in Tfh cells that are essential for GC formation and suggest that BCL6 helps maintain Tfh cell phenotypes in a T cell non-autonomous manner.

Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes.

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.

Inhibiting Inflammation with Myeloid Cell-Specific Nanobiologics Promotes Organ Transplant Acceptance.

Inducing graft acceptance without chronic immunosuppression remains an elusive goal in organ transplantation. Using an experimental transplantation mouse model, we demonstrate that local macrophage activation through dectin-1 and toll-like receptor 4 (TLR4) drives trained immunity-associated cytokine production during allograft rejection. We conducted nanoimmunotherapeutic studies and found that a short-term mTOR-specific high-density lipoprotein (HDL) nanobiologic treatment (mTORi-HDL) averted macrophage aerobic glycolysis and the epigenetic modifications underlying inflammatory cytokine production. The resulting regulatory macrophages prevented alloreactive CD8+ T cell-mediated immunity and promoted tolerogenic CD4+ regulatory T (Treg) cell expansion. To enhance therapeutic efficacy, we complemented the mTORi-HDL treatment with a CD40-TRAF6-specific nanobiologic (TRAF6i-HDL) that inhibits co-stimulation. This synergistic nanoimmunotherapy resulted in indefinite allograft survival. Together, we show that HDL-based nanoimmunotherapy can be employed to control macrophage function in vivo. Our strategy, focused on preventing inflammatory innate immune responses, provides a framework for developing targeted therapies that promote immunological tolerance.

B Cell Receptor and CD40 Signaling Are Rewired for Synergistic Induction of the c-Myc Transcription Factor in Germinal Center B Cells.

Positive selection of germinal center (GC) B cells is driven by B cell receptor (BCR) affinity and requires help from follicular T helper cells. The transcription factors c-Myc and Foxo1 are critical for GC B cell selection and survival. However, how different affinity-related signaling events control these transcription factors in a manner that links to selection is unknown. Here we showed that GC B cells reprogram CD40 and BCR signaling to transduce via NF-κB and Foxo1, respectively, whereas naive B cells propagate both signals downstream of either receptor. Although either BCR or CD40 ligation induced c-Myc in naive B cells, both signals were required to highly induce c-Myc, a critical mediator of GC B cell survival and cell cycle reentry. Thus, GC B cells rewire their signaling to enhance selection stringency via a requirement for both antigen receptor- and T cell-mediated signals to induce mediators of positive selection.

Intratumoral NKT cell accumulation promotes antitumor immunity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a potentially lethal disease lacking effective treatments. Its immunosuppressive tumor microenvironment (TME) allows it to evade host immunosurveillance and limits response to immunotherapy. Here, using the mouse KRT19-deficient (sgKRT19-edited) PDA model, we find that intratumoral accumulation of natural killer T (NKT) cells is required to establish an immunologically active TME. Mechanistically, intratumoral NKT cells facilitate type I interferon (IFN) production to initiate an antitumor adaptive immune response, and orchestrate the intratumoral infiltration of T cells, dendritic cells, natural killer cells, and myeloid-derived suppressor cells. At the molecular level, NKT cells promote the production of type I IFN through the interaction of their CD40L with CD40 on myeloid cells. To evaluate the therapeutic potential of these observations, we find that administration of folinic acid to mice bearing PDA increases NKT cells in the TME and improves their response to anti-PD-1 antibody treatment. In conclusion, NKT cells have an essential role in the immune response to mouse PDA and are potential targets for immunotherapy.

B cell peripheral tolerance is promoted by cathepsin B protease.

B cells that bind soluble autoantigens receive chronic signaling via the B cell receptor (signal-1) in the absence of strong costimulatory signals (signal-2), and this leads to their elimination in peripheral tissues. The factors determining the extent of soluble autoantigen-binding B cell elimination are not fully understood. Here we demonstrate that the elimination of B cells chronically exposed to signal-1 is promoted by cathepsin B (Ctsb). Using hen egg lysozyme-specific (HEL-specific) immunoglobulin transgenic (MD4) B cells and mice harboring circulating HEL, we found improved survival and increased proliferation of HEL-binding B cells in Ctsb-deficient mice. Bone marrow chimera experiments established that both hematopoietic and nonhematopoietic sources of Ctsb were sufficient to promote peripheral B cell deletion. The depletion of CD4+ T cells overcame the survival and growth advantage provided by Ctsb deficiency, as did blocking CD40L or removing CD40 from the chronically antigen-engaged B cells. Thus, we suggest that Ctsb acts extracellularly to reduce soluble autoantigen-binding B cell survival and that its actions restrain CD40L-dependent pro-survival effects. These findings identify a role for cell-extrinsic protease activity in establishing a peripheral self-tolerance checkpoint.

IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer.

CD40 is a central costimulatory receptor implicated in productive antitumor immune responses across multiple cancers, including bladder cancer. Despite strong preclinical rationale, systemic administration of therapeutic agonistic antibodies targeting the CD40 pathway has demonstrated dose-limiting toxicities with minimal clinical activity, emphasizing an important need for optimized CD40-targeted approaches, including rational combination therapy strategies. Here, we describe a role for the endogenous IL-15 pathway in contributing to the therapeutic activity of CD40 agonism in orthotopic bladder tumors, with upregulation of transpresented IL-15/IL-15Rα surface complexes, particularly by cross-presenting conventional type 1 DCs (Dendritic Cells), and associated enrichment of activated CD8 T cells. In bladder cancer patient samples, we identify DCs as the primary source of IL-15, although they lack high levels of IL-15Rα at baseline. Using humanized immunocompetent orthotopic bladder tumor models, we demonstrate the ability to therapeutically augment this interaction through combined treatment with anti-CD40 agonist antibodies and exogenous IL-15, including the fully-human Fc-optimized antibody 2141-V11 currently in clinical development for the treatment of bladder cancer. Collectively, these data reveal an important role for IL-15 in mediating antitumor CD40 agonist responses in bladder cancer and provide key proof-of-concept for combined use of Fc-optimized anti-CD40 agonist antibodies and agents targeting the IL-15 pathway. These data support expansion of ongoing clinical studies evaluating anti-CD40 agonist antibodies and IL-15-based approaches to develop combinations of these promising therapeutics for the treatment of patients with bladder cancer.

Humoral responses are enhanced by facilitating B cell viability by Fcrl5 overexpression in B cells.

B cell initial activity is regulated through a balance of activation and suppression mediated by regulatory molecules expressed in B cells; however, the molecular mechanisms underlying this process remain incompletely understood. In this study, we investigated the function of the Fc receptor-like (Fcrl) family molecule Fcrl5, which is constitutively expressed in naive B cells, in humoral immune responses. Our study demonstrated that B cell-specific overexpression of Fcrl5 enhanced antibody (Ab) production in both T cell-independent type 1 (TI1) and T cell-dependent (TD) responses. Additionally, it promoted effector B cell formation under competitive conditions in TD responses. Mechanistically, in vitro ligation of Fcrl5 by agonistic Abs reduced cell death and enhanced proliferation in lipopolysaccharide-stimulated B cells. In the presence of anti-CD40 Abs and IL-5, the Fcrl5 ligation not only suppressed cell death but also enhanced differentiation into plasma cells. These findings reveal a novel role of Fcrl5 in promoting humoral immune responses by enhancing B cell viability and plasma cell differentiation.

Lighting up the tumor fire with low-dose irradiation.

Current efforts combining immunotherapy and radiation have focused on high-dose radiation delivered to few tumor lesions, aiming to generate diffuse abscopal effects; however, these effects are uncommon in patients. Three recent studies in mouse tumor models and human cancer patients show that low-dose radiation (LDRT) delivered to all tumor lesions effectively mobilizes innate and adaptive immunity and synergizes with immunotherapy. These new findings suggest LDRT's potential as an immune amplifier capable of reprogramming the tumor microenvironment, instigating inflammation, and sensitizing 'cold' tumors to immune checkpoint blockade responsiveness.

CD40-CD154: A perspective from type 2 immunity.

The interaction between CD40 and CD154 (CD40 ligand) is central in immunology, participating in CD4+ T cell priming by dendritic cells (DC), CD4+ T cell help to B cells and classical macrophage activation by CD4+ T cells. However, its role in the Th2 side of immunology including helminth infection remains incompletely understood. Contrary to viral and bacterial stimuli, helminth products usually do not cause CD40 up-regulation in DC, and exogenous CD40 ligation drives Th2-biased systems towards Th1. On the other hand, CD40 and CD154 are necessary for induction of most Th2 responses. We attempt to reconcile these observations, mainly by proposing that (i) CD40 up-regulation in DC in Th2 systems is mostly induced by alarmins, (ii) the Th2 to Th1 shift induced by exogenous CD40 ligation is related to the capacity of such ligation to enhance IL-12 production by myeloid cells, and (iii) signals elicited by endogenous CD154 available in Th2 contexts and by exogenous CD40 ligation are probably different. We stress that CD40-CD154 is important beyond cognate cellular interactions. In such a context, we argue that the proliferation response of B-cells to IL-4 plus CD154 reflects a Th2-specific mechanism for polyclonal B-cell amplification and IgE production at infection sites. Finally, we argue that CD154 is a general immune activation signal across immune polarization including Th2, and propose that competition for CD154 at tissue sites may provide negative feedback on response induction at each site.

CD40 Expression by B cells is Required for Optimal Immunity to Murine Pneumocystis Infection.

CD40-CD40L interactions are critical for controlling Pneumocystis infection. However, which CD40-expressing cell populations are important for this interaction have not been well-defined. We used a cohousing mouse model of Pneumocystis infection, combined with flow cytometry and qPCR, to examine the ability of different populations of cells from C57BL/6 mice to reconstitute immunity in CD40 knockout (KO) mice. Unfractionated splenocytes, as well as purified B cells, were able to control Pneumocystis infection, while B cell depleted splenocytes and unstimulated bone-marrow derived dendritic cells (BMDCs) were unable to control infection in CD40 KO mice. Pneumocystis antigen-pulsed BMDCs showed early, but limited, control of infection. Consistent with recent studies that have suggested a role for antigen presentation by B cells, using cells from immunized animals, B cells were able to present Pneumocystis antigens to induce proliferation of T cells. Thus, CD40 expression by B cells appears necessary for robust immunity to Pneumocystis.

Platelet transfusion enhances pro-aggregatory status shortly after coronary artery bypass grafting (CABG while modulating platelet pro-inflammatory state 1-week post-surgery.

During coronary artery bypass grafting (CABG), the surgical procedure, particularly the manipulation of the major arteries of the heart, induces a significant inflammatory state that may compromise platelet function to the extent that platelet transfusion is required. Given stored platelets as a major source of biological mediators, this study investigates the effects of platelet transfusion on the major pro-aggregatory, pro-inflammatory and immunomodulatory markers of platelets. Platelets from 20 patients, 10 who received platelet transfusion and 10 without, were subjected to flow cytometery where P-selectin and CD40 ligand (CD40L) expressions and PAC-1 binding (activation-specific anti GPIIb/GPIIIa antibody) analysed at five-time points of 24 h before surgery, immediately, 2 h, 24 h and 1 week after surgery. Analysis of intra-platelet transforming growth factor-beta-1 (TGF-ß1) was also conducted using western blotting. Patients with platelet transfusion showed increased levels of P-selectin, CD40L and intra-platelet TGF-ß1 2-h after surgery compared to those without transfusion (p < 0.05). PAC-1 binding was increased 24 h after surgery in transfused patients (p < 0.05). Given the significant post-transfusion elevation of platelet TGF-ß1, P-sel/CD40L reduction in transfused patients a week after was of much interest. This study showed for the first time the significant effects of platelet transfusion on the pro-inflammatory, pro-aggeregatory and immunomodulatory state of platelets in CABG patients, which manifested with immediate, midterm and delayed consequences. While the increased pro-inflammatory conditions manifested as an immediate effect of platelet transfusion, the pro-aggregatory circumstances emerged 24 h post-transfusion. A week after surgery, attenuations of pro-inflammatory markers of platelets in transfused patients were shown, which might be due to the immunomodulatory effects of TGF-ß1.

Modulating CD40 and integrin signaling in the proinflammatory nexus using a 15-amino-acid peptide, KGYY15.

CD40 signaling has long been a target in autoimmunity. Attempts to block signaling between CD40 and CD154 during clinical trials using monoclonal antibodies suffered severe adverse events. Previously, we developed a peptide, KGYY15, that targets CD40 and, in preclinical trials, prevents type 1 diabetes in >90% of cases and reverses new-onset hyperglycemia in 56% of cases. It did so by establishing normal effector T-cell levels rather than ablating the cells and causing immunosuppression. However, the relationship between KGYY15 and other elements of the complex signaling network of CD40 is not clear. Studying interactions between proteins from autoimmune and nonautoimmune mice, we demonstrate interactions between CD40 and integrin CD11a/CD18, which complicates the understanding of the inflammatory nexus and how to prevent autoinflammation. In addition to interacting with CD40, KGYY15 interacts with the integrins CD11a/CD18 and CD11b/CD18. We argue that modulation of CD40-CD154 signaling may be more advantageous than complete inhibition because it may preserve normal immunity to pathogens.

Fibroblast-like synoviocytes preferentially induce terminal differentiation of IgD+ memory B cells instead of naïve B cells.

Rheumatoid arthritis (RA) is a systemic autoimmune disease driven by highly active autoantibody-producing B cells. Activation of B cells is maintained within ectopic germinal centres found in affected joints. Fibroblast-like synoviocytes (FLS) present in inflamed joints support B-cell survival, activation, and differentiation. CD27+ memory B cells and naive B cells show very different responses to activation, particularly by CD40 ligand (CD40L). We show that FLS-dependent activation of human B cells is dependent on interleukin-6 (IL-6) and CD40L. FLS have been shown to activate both naive and memory B cells. Whether the activating potential of FLS is different for naive and memory B cells has not been investigated. Our results suggest that FLS-induced activation of B cells is dependent on IL-6 and CD40L. While FLS are able to induce plasma cell differentiation, isotype switching, and antibody production in memory B cells, the ability of FLS to activate naive B cells is significantly lower.