Clonal Deletion of Tumor-Specific T Cells by Interferon-γ Confers Therapeutic Resistance to Combination Immune Checkpoint Blockade.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

Treatment with a CD40 Antagonist Antibody Reverses Severe Proteinuria and Loss of Saliva Production and Restores Glomerular Morphology in Murine Systemic Lupus Erythematosus.

CD40 is a costimulatory receptor on APCs that is critical for the induction and maintenance of humoral and cell-mediated immunity. Accordingly, CD40 and its ligand, CD40L, have long been considered targets for the treatment of autoimmune diseases. We developed a rat/mouse chimeric anti-mouse CD40 antagonist mAb, 201A3, and evaluated its ability to alleviate murine lupus. Treatment of NZB/W-F1 mice with 201A3 after the onset of severe proteinuria rapidly reversed established severe proteinuria and nephritis and largely restored normal glomerular and tubular morphology. This coincided with a normalization of the expression of genes associated with proteinuria and injury by kidney parenchymal cells. Anti-CD40 treatment also prevented and reversed loss of saliva production and sialadenitis. These effects on kidney and salivary gland function were confirmed using mice of a second strain, MRL/Mp-lpr/lpr, and extended to alleviating joint inflammation. Immunologically, anti-CD40 treatment disrupted multiple processes that contribute to the pathogenesis of systemic lupus erythematosus (SLE), including autoreactive B cell activation, T effector cell function in target tissues, and type I IFN production. This ability to disrupt disease-critical immunological mechanisms, to reverse glomerular and tubular injury at the cellular and gene expression levels, and to confer exceptional therapeutic efficacy suggests that CD40 is a central disease pathway in murine SLE. Thus, a CD40 antagonist Ab could be an effective therapeutic in the treatment of SLE.

A novel allele for inducible Cre expression in germinal center B cells.

The germinal center reaction is essential for efficient humoral immunity, but it can also give rise to B cell lymphomas. Cre/loxP-mediated conditional gene knock-out or knock-in can be used for the genetic manipulation of germinal center B cells in vivo. Here we present a novel allele, Cγ1-CreERT2, that allows for timed activation of Cre recombinase in a small fraction of germinal center B cells. This allele will be useful to study normal and malignant germinal center B cell development in vivo.

IL-36 receptor antagonistic antibodies inhibit inflammatory responses in preclinical models of psoriasiform dermatitis.

Psoriasis vulgaris (PV) results from activation of IL-23/Th17 immune pathway and is further amplified by cytokines/chemokines from skin cells. Among skin-derived pro-inflammatory cytokines, IL-36 family members are highly upregulated in PV patients and play a critical role in general pustular psoriasis. However, there is limited data showing crosstalk between the IL-23 and IL-36 pathways in PV. Herein, potential attenuation of skin inflammation in the IL-23-induced mouse model of psoriasiform dermatitis by functional inhibition of IL-36 receptor (IL-36R) was interrogated. Anti-mouse IL-36R monoclonal antibodies (mAbs) were generated and validated in vitro by inhibiting IL-36α-induced secretion of CXCL1 from NIH 3T3 cells. Antibody target engagement was demonstrated by inhibition of CXCL1 production in a novel acute model of IL-36α systemic injection in mice. In addition, anti-IL-36R mAbs inhibited tissue inflammation and inflammatory gene expression in an IL-36α ear injection model of psoriasiform dermatitis demonstrating engagement of the target in the ear skin. To elucidate the possible role of IL-36 signalling in IL-23/Th17 pathway, the ability of anti-IL-36R mAbs to inhibit skin inflammation in an IL-23 ear injection model was assessed. Inhibiting the IL-36 pathway resulted in significant attenuation of skin thickening and psoriasis-relevant gene expression. Taken together, these data suggest a role for IL-36 signalling in the IL-23/Th17 signalling axis in PV.

Fas receptor expression in germinal-center B cells is essential for T and B lymphocyte homeostasis.

Fas is highly expressed in activated and germinal center (GC) B cells but can potentially be inactivated by misguided somatic hypermutation. We employed conditional Fas-deficient mice to investigate the physiological functions of Fas in various B cell subsets. B cell-specific Fas-deficient mice developed fatal lymphoproliferation due to activation of B cells and T cells. Ablation of Fas specifically in GC B cells reproduced the phenotype, indicating that the lymphoproliferation initiates in the GC environment. B cell-specific Fas-deficient mice also showed an accumulation of IgG1(+) memory B cells expressing high amounts of CD80 and the expansion of CD28-expressing CD4(+) Th cells. Blocking T cell-B cell interaction and GC formation completely prevented the fatal lymphoproliferation. Thus, Fas-mediated selection of GC B cells and the resulting memory B cell compartment is essential for maintaining the homeostasis of both T and B lymphocytes.

IgG1 B cell receptor signaling is inhibited by CD22 and promotes the development of B cells whose survival is less dependent on Ig alpha/beta.

We describe a mouse strain in which B cell development relies either on the expression of membrane-bound immunoglobulin (Ig) gamma1 or mu heavy chains. Progenitor cells expressing gamma1 chains from the beginning generate a peripheral B cell compartment of normal size with all subsets, but a partial block is seen at the pro- to pre-B cell transition. Accordingly, gamma1-driven B cell development is disfavored in competition with developing B cells expressing a wild-type (WT) IgH locus. However, the mutant B cells display a long half-life and accumulate in the mature B cell compartment, and even though partial truncation of the Ig alpha cytoplasmic tail compromises their development, it does not affect their maintenance, as it does in WT cells. IgG1-expressing B cells showed an enhanced Ca(2+) response upon B cell receptor cross-linking, which was not due to a lack of inhibition by CD22. The enhanced Ca(2+) response was also observed in mature B cells that had been switched from IgM to IgG1 expression in vivo. Collectively, these results suggest that the gamma1 chain can exert a unique signaling function that can partially replace that of the Ig alpha/beta heterodimer in B cell maintenance and may contribute to memory B cell physiology.

Cytoplasmic Ig alpha serine/threonines fine-tune Ig alpha tyrosine phosphorylation and limit bone marrow plasma cell formation.

Igα serine 191 and 197 and threonine 203, which are located in proximity of the Igα ITAM, dampen Igα ITAM tyrosine phosphorylation. In this study, we show that mice with targeted mutations of Igα S191, 197, and T203 displayed elevated serum IgG2c and IgG2b concentrations and had elevated numbers of IgG2c- and IgG2b-secreting cells in the bone marrow. BCR-induced Igα tyrosine phosphorylation was slightly increased in splenic B cells. Our results suggest that Igα serine/threonines limit formation of IgG2c- and IgG2b-secreting bone marrow plasma cells, possibly by fine-tuning Igα tyrosine-mediated BCR signaling.

Antiviral Drug Discovery for the Treatment of COVID-19 Infections.

The coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a recently emerged human coronavirus. COVID-19 vaccines have proven to be successful in protecting the vaccinated from infection, reducing the severity of disease, and deterring the transmission of infection. However, COVID-19 vaccination faces many challenges, such as the decline in vaccine-induced immunity over time, and the decrease in potency against some SARS-CoV-2 variants including the recently emerged Omicron variant, resulting in breakthrough infections. The challenges that COVID-19 vaccination is facing highlight the importance of the discovery of antivirals to serve as another means to tackle the pandemic. To date, neutralizing antibodies that block viral entry by targeting the viral spike protein make up the largest class of antivirals that has received US FDA emergency use authorization (EUA) for COVID-19 treatment. In addition to the spike protein, other key targets for the discovery of direct-acting antivirals include viral enzymes that are essential for SARS-CoV-2 replication, such as RNA-dependent RNA polymerase and proteases, as judged by US FDA approval for remdesivir, and EUA for Paxlovid (nirmatrelvir + ritonavir) for treating COVID-19 infections. This review presents an overview of the current status and future direction of antiviral drug discovery for treating SARS-CoV-2 infections, covering important antiviral targets such as the viral spike protein, non-structural protein (nsp) 3 papain-like protease, nsp5 main protease, and the nsp12/nsp7/nsp8 RNA-dependent RNA polymerase complex.

Structural basis for continued antibody evasion by the SARS-CoV-2 receptor binding domain.

Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.

A novel recombinant fusion protein encoding a 20-amino acid residue of the third extracellular (E3) domain of CCR2 neutralizes the biological activity of CCL2.

CCL2 is a key CC chemokine that has been implicated in a variety of inflammatory autoimmune diseases and in tumor progression and it is therefore an important target for therapeutic intervention in these diseases. Soluble receptor-based therapy is a known approach for neutralizing the in vivo functions of soluble mediators. Owing to the complexity of seven-transmembrane G protein-coupled receptors, efforts to generate neutralizing soluble chemokine receptors have so far failed. We developed a strategy that is based on the generation of short recombinant proteins encoding different segments of a G protein-coupled receptor, and tested the ability of each of them to bind and neutralize its target chemokine. We show that a fusion protein comprised of as few as 20 aa of the third extracellular (E3) domain of the CCL2 receptor, stabilized by the IgG H chain Fc domain (E3-IgG or BL-2030), selectively binds CCL2 and CCL16 and effectively neutralizes their biological activities. More importantly, E3-IgG (BL-2030) could effectively suppress the in vivo biological activity of CCL2, attenuating ongoing experimental autoimmune encephalomyelitis, as well as the development of human prostate tumor in SCID mice.