IKKalpha-Mediated Non-canonical NF-kappaB Signaling is Required to Support Murine Gammaherpesvirus 68 Latency In Vivo

Non-canonical NF-kappaB signaling is activated in B cells via TNF receptor superfamily members CD40, Lymphotoxin beta-R, and BAFF-R. The non-canonical pathway is required at multiple stages of B-cell maturation and differentiation, including the germinal center reaction. However, the role of this pathway in gammaherpesvirus latency is not well understood. Murine gammaherpesvirus 68 (MHV68) is a genetically tractable system used to define pathogenic determinants. Mice lacking the BAFF-R exhibit defects in splenic follicle formation and are greatly reduced for MHV68 latency. We report a novel approach to disrupt non-canonical NF-kappaB signaling exclusively in cells infected with MHV68. We engineered a recombinant virus that expresses a dominant negative form of IKKalpha, named IKKα-SA, with S176A and S180A mutations that prevent phosphorylation by NIK. We controlled for the transgene insertion by introducing two all-frame stop codons into the IKKα-SA gene. The IKKα-SA mutant but not the IKKα-SA.STOP control virus impaired LTbetaR-mediated activation of NF-kappaB p52 upon fibroblast infection. IKKα-SA expression did not impact replication in primary fibroblasts or in the lungs of mice following intranasal inoculation. However, the IKKα-SA mutant was severely defective in colonization of the spleen and in the establishment of latency compared to the IKKα-SA.STOP control and WT MHV68 at 16 dpi. Reactivation was undetectable in splenocytes infected with the IKKα-SA mutant, but reactivation in peritoneal cells was not impacted by IKKα-SA. Taken together, the non-canonical NF-kappaB signaling pathway is essential for the establishment of latency in the secondary lymphoid organs of mice infected with the murine gammaherpesvirus pathogen MHV68.

PHGDH is required for germinal center formation and is a therapeutic target in MYC-driven lymphoma

The synthesis of serine from glucose is a key metabolic pathway supporting cellular proliferation in healthy and malignant cells. Despite this, the role that this aspect of metabolism plays in germinal center biology and pathology is not known. Here, we performed a comprehensive characterization of the role of the serine synthesis pathway in germinal center B cells and lymphomas derived from these cells. We demonstrate that upregulation of a functional serine synthesis pathway is a metabolic hallmark of B-cell activation and the germinal center reaction. Inhibition of phosphoglycerate dehydrogenase (PHGDH), the first and rate limiting enzyme in this pathway, leads to defective germinal formation and impaired high-affinity antibody production. In addition, overexpression of enzymes involved in serine synthesis is a characteristic of germinal center B-cell derived lymphomas, with high levels of expression being predictive of reduced overall survival in diffuse large B cell lymphoma. Inhibition of PHGDH induces apoptosis in lymphoma cells reducing disease progression. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in lymphoma.

Aging-Associated Epigenetic Reprogramming Alters the Germinal Center Reaction and Targets Pathways Related to Lymphomagenesis

Aging is associated with suboptimal germinal center (GC) responses and inferior outcomes of GC-derived lymphomas such as diffuse large B-cell lymphoma (DLBCL). At the molecular level, aging is characterized by epigenetic alterations of DNA CpG methylation and chromatin architecture that ultimately affect cellular functions. The B-cell/T-cell immune synapse during the transitional stages of the GC is governed by a series of epigenetic switches. Frequent mutations in DLBCL directly impact epigenetic regulators and signaling pathways in the immune synapse. Here, we investigated the impact of aging-associated epigenetic alterations in the functionality of the GC response in parallel with biological characteristics of DLBCL in the elderly. We conducted an integrative characterization of epigenomic, transcriptomic and phenotypic changes of B and T cells during the GC reaction in younger (3-4 months) vs. older (21-23 months) mice by immunophenotyping (flow cytometry), DNA methylation sequencing of sorted sub-populations, single cell (SC) RNA-sequencing (coding and BCR/TCR) and SC-multiomics (integration of RNA-sequencing and ATAC-sequencing). We also performed DNA methylation sequencing, RNA-sequencing and NGS in a cohort of 266 DLBCL including 51 pts >75 y.o. In agreement with reported phenotypes in elderly humans (Collier D. et al., Nature, 2021), the GC reaction in older mice was characterized by a significative reduction in IgG3+ (p=0.0001) and increased in IgM+ (p=0.009) B cells (FAS+ CD38-); whereas the non-GC B cell compartment (FAS- CD38+) displayed an increase in age-associated B cells (ABCs, p=0.0001) and reduction in follicular B-cells (p=0.004). Furthermore, older mice displayed an expansion of a FAS+CD38+ population of B cells (p=0.0001). Regarding T cells, we observed a global reduction in CD4+ (p=0.01) but not in CD8+ cells; however, older mice showed an expansion of PD1+/CD4+ and PD1+/CD8+ T cells (p=0.0004 and p=0.0003, respectively). Furthermore, older mice displayed increased TFH (p=0.0001), Tregs (p=0001) and ICOS+ Treg (p=0001) populations. SC-RNA-seq of B and T cells validated these alterations and identified transcriptionally-defined sub-populations including expansion of clonal GZMK CD8+ TOX+ T cells (TAA cells) and activation of "cytokine production" in T cells (FDR=3.01e-27), both phenotypes associated with "inflammaging" (Mogilenko et al., Immunity, 2020). Epigenetic changes in older B-cells showed aberrant hypermethylation of gene promoters and hypomethylation of intergenic regions including enhancers, resulting in changes of chromatin architecture and gene expression. In GC B-cells but not naïve B-cells (NBC), genes whose promoter was aberrantly hypermethylated in older mice were enriched for targets of SUZ12 (FDR= 5.1e -12), EZH2 (FDR= 5.1e -12) and JARID2 (FDR= 6.8e -10), key components of the PRC2 complex. Older mice B-cells also displayed decreased chromatin accessibility in genes functionally enriched for "semaphorin-plexin signaling pathway" (FDR=5.3e -03) which regulates TFH/B-cell immune synapse as well as decreased expression of linker histone H1 isoforms B-E in GCB cells (q=0.0006; q=0.0003; q=0.008, q=4.64E-10, respectively). Notably, the age-associated reprogramming observed in the germinal center reaction globally targets pro-tumorigenic pathways that are comparably altered in elderly DLBCL. We observed that older pts (>75 y.o.) vs. younger pts (55-65 y.o.) had increased hypermethylation of gene promoters and hypomethylation of intergenic regions with deregulation of epigenetic modifiers (including PCR2 members) and immune synapse genes (including BCR signaling). There were no differences in the prevalence of specific mutations between these two populations. However, DLBCL in elderly are more frequently of the ABC subtype (~60%, p<0.05) and presented with an inflammatory tumor microenvironment (41% vs 27% in younger DLBCL, p<0.001, corrected by COO) that is characterized by increased infiltration of macrophages (p=0.001), Tregs (p=0.001) and CD8+ PD1 highcells (p=0.001), phenocopying changes observed in the germinal center of older mice. Thus, age-associated epigenetic reprogramming results in perturbation of pathways regulating the B-cell/T-cell immune synapse during the normal germinal center reaction and may underlie some of the biological characteristics observed in elderly DLBCL patients. Disclosures Lara-Garcia: Johnson and Johnson: Current holder of individual stocks in a privately-held company; Bristol Myers Squibb: Divested equity in a private or publicly-traded company in the past 24 months; Moderna: Divested equity in a private or publicly-traded company in the past 24 months; Pfizer: Divested equity in a private or publicly-traded company in the past 24 months; Regeneron: Divested equity in a private or publicly-traded company in the past 24 months; Merck: Divested equity in a private or publicly-traded company in the past 24 months; Fortress Biotech: Divested equity in a private or publicly-traded company in the past 24 months. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

The germinal center reaction depends on RNA methylation and divergent functions of specific methyl readers

Long-lasting immunity depends on the generation of protective antibodies through the germinal center (GC) reaction. N6-methyladenosine (m6A) modification of mRNAs by METTL3 activity modulates transcript lifetime primarily through the function of m6A readers; however, the physiological role of this molecular machinery in the GC remains unknown. Here, we show that m6A modifications by METTL3 are required for GC maintenance through the differential functions of m6A readers. Mettl3-deficient GC B cells exhibited reduced cell-cycle progression and decreased expression of proliferation- and oxidative phosphorylation–related genes. The m6A binder, IGF2BP3, was required for stabilization of Myc mRNA and expression of its target genes, whereas the m6A reader, YTHDF2, indirectly regulated the expression of the oxidative phosphorylation gene program. Our findings demonstrate how two independent gene networks that support critical GC functions are modulated by m6A through distinct mRNA binders.

The Role of Coinfections in the EBV–Host Broken Equilibrium

The Epstein–Barr virus (EBV) is a well-adapted human virus, and its infection is exclusive to our species, generally beginning in the childhood and then persisting throughout the life of most of the affected adults. Although this infection generally remains asymptomatic, EBV can trigger life-threatening conditions under unclear circumstances. The EBV lifecycle is characterized by interactions with other viruses or bacteria, which increases the probability of awakening its pathobiont capacity. For instance, EBV infects B cells with the potential to alter the germinal center reaction (GCR)—an adaptive immune structure wherein mutagenic-driven processes take place. HIV- and Plasmodium falciparum-induced B cell hyperactivation also feeds the GCR. These agents, along with the B cell tropic KSHV, converge in the ontogeny of germinal center (GC) or post-GC lymphomas. EBV oral transmission facilitates interactions with local bacteria and HPV, thereby increasing the risk of periodontal diseases and head and neck carcinomas. It is less clear as to how EBV is localized in the stomach, but together with Helicobacter pylori, they are known to be responsible for gastric cancer. Perhaps this mechanism is reminiscent of the local inflammation that attracts different herpesviruses and enhances graft damage and chances of rejection in transplanted patients. In this review, we discussed the existing evidence suggestive of EBV possessing the potential to synergize or cooperate with these agents to trigger or worsen the disease.

Genome-wide mutational signatures of immunological diversification in normal lymphocytes

A lymphocyte suffers many threats to its genome, including programmed mutation during differentiation, antigen-driven proliferation and residency in diverse microenvironments. After developing protocols for single-cell lymphocyte expansions, we sequenced whole genomes from 717 normal naive and memory B and T lymphocytes and hematopoietic stem cells. Lymphocytes carried more point mutations and structural variation than stem cells, accruing at higher rates in T than B cells, attributable to both exogenous and endogenous mutational processes. Ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every one on-target IGV mutation during the germinal center reaction. Structural variation was 16-fold higher in lymphocytes than stem cells, with ~15% of deletions being attributable to off-target RAG activity.