Sequencing of Kaposi's Sarcoma Herpesvirus (KSHV) genomes from persons of diverse ethnicities and provenances with KSHV-associated diseases demonstrate multiple infections, novel polymorphisms, and low intra-host variance.

Recently published near full-length KSHV genomes from a Cameroon Kaposi sarcoma case-control study showed strong evidence of viral recombination and mixed infections, but no sequence variations associated with disease. Using the same methodology, an additional 102 KSHV genomes from 76 individuals with KSHV-associated diseases have been sequenced. Diagnoses comprise all KSHV-associated diseases (KAD): Kaposi sarcoma (KS), primary effusion lymphoma (PEL), KSHV-associated large cell lymphoma (KSHV-LCL), a type of multicentric Castleman disease (KSHV-MCD), and KSHV inflammatory cytokine syndrome (KICS). Participants originated from 22 different countries, providing the opportunity to obtain new near full-length sequences of a wide diversity of KSHV genomes. These include near full-length sequence of genomes with KSHV K1 subtypes A, B, C, and F as well as subtype E, for which no full sequence was previously available. High levels of recombination were observed. Fourteen individuals (18%) showed evidence of infection with multiple KSHV variants (from two to four unique genomes). Twenty-six comparisons of sequences, obtained from various sampling sites including PBMC, tissue biopsies, oral fluids, and effusions in the same participants, identified near complete genome conservation between different biological compartments. Polymorphisms were identified in coding and non-coding regions, including indels in the K3 and K15 genes and sequence inversions here reported for the first time. One such polymorphism in KSHV ORF46, specific to the KSHV K1 subtype E2, encoded a mutation in the leucine loop extension of the uracil DNA glycosylase that results in alteration of biochemical functions of this protein. This confirms that KSHV sequence variations can have functional consequences warranting further investigation. This study represents the largest and most diverse analysis of KSHV genome sequences to date among individuals with KAD and provides important new information on global KSHV genomics.

UNG-RPA interaction governs the choice between high-fidelity and mutagenic uracil repair.

Mammalian Uracil DNA glycosylase (UNG) removes uracils and initiates high-fidelity base excision repair to maintain genomic stability. During B cell development, activation-induced cytidine deaminase (AID) creates uracils that UNG processes in an error-prone fashion to accomplish immunoglobulin (Ig) somatic hypermutation (SHM) or class switch recombination (CSR). The mechanism that governs high-fidelity versus mutagenic uracil repair is not understood. The B cell tropic gammaherpesvirus (GHV) encodes a functional homolog of UNG that can process AID induced genomic uracils. GHVUNG does not support hypermutation, suggesting intrinsic properties of UNG influence repair outcome. Noting the structural divergence between the UNGs, we define the RPA interacting motif as the determinant of mutation outcome. UNG or RPA mutants unable to interact with each other, only support high-fidelity repair. In B cells, transversions at the Ig variable region are abated while CSR is supported. Thus UNG-RPA governs the generation of mutations and has implications for locus specific mutagenesis in B cells and deamination associated mutational signatures in cancer.

Acid-active proteases to optimize dietary protein digestibility: a step towards sustainable nutrition.

Introduction: Historically, prioritizing abundant food production often resulted in overlooking nutrient quality and bioavailability, however, environmental concerns have now propelled sustainable nutrition and health efficacy to the forefront of global attention. In fact, increasing demand for protein is the major challenge facing the food system in the 21st century with an estimation that 70% more food is needed by 2050. This shift has spurred interest in plant-based proteins for their sustainability and health benefits, but most alternative sources of protein are poorly digestible. There are two approaches to solve digestibility: improve the digestibility of food proteins or improve the digestive capacity of consumers. Enhancing nutrient digestibility and bioavailability across diverse protein sources is crucial, with proteases presenting a promising avenue. Research, inspired by the proteases of human breast milk, has demonstrated that exogenous microbial proteases can activate within the human digestive tract and substantially increase the digestion of targeted proteins that are otherwise difficult to fully digest. Methods: Here, we introduce the use of an acid-active family of bacterial proteases (S53) to improve the digestibility and nutritional quality of a variety of protein sources, evaluated using the INFOGEST 2.0 protocol. Results: Results from in vitro digestibility indicate that the most effective protease in the S53 family substantially improves the digestibility of an array of animal and plant-derived proteins-soy, pea, chickpea, rice, casein, and whey. On average, this protease elevated protein digestibility by 115% during the gastric phase and by 15% in the intestinal phase, based on the degree of hydrolysis. Discussion: The widespread adoption of these proteases has the potential to enhance nutritional value and contribute to food security and sustainability. This approach would complement ongoing efforts to improve proteins in the food supply, increase the quality of more sustainable protein sources and aid in the nourishment of patients with clinically compromised, fragile intestines and individuals like older adults and high-performance athletes who have elevated protein needs.

Multifaceted roles for STAT3 in gammaherpesvirus latency revealed through in vivo B cell knockout models.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). To better understand the role of STAT3 during gammaherpesvirus latency and the B cell response to infection, we used the model pathogen murine gammaherpesvirus 68 (MHV68). Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak MHV68 latency approximately sevenfold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to wild-type (WT) littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeric mice consisting of WT and STAT3 knockout B cells. We discovered a dramatic reduction in latency in STAT3 knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that MHV68 infection shifts the gene signature toward proliferation and away from type I and type II IFN responses. Loss of STAT3 largely reversed the virus-driven transcriptional shift without impacting the viral gene expression program. STAT3 promoted B cell processes of the germinal center, including IL-21-stimulated downregulation of surface CD23 on B cells infected with MHV68 or EBV. Together, our data provide mechanistic insights into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.IMPORTANCEThere are no directed therapies to the latency program of the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus. Activated host factor signal transducer and activator of transcription 3 (STAT3) is a hallmark of cancers caused by these viruses. We applied the murine gammaherpesvirus pathogen system to explore STAT3 function upon primary B cell infection in the host. Since STAT3 deletion in all CD19+ B cells of infected mice led to altered B and T cell responses, we generated chimeric mice with both normal and STAT3-deleted B cells. B cells lacking STAT3 failed to support virus latency compared to normal B cells from the same infected animal. Loss of STAT3 impaired B cell proliferation and differentiation and led to a striking upregulation of interferon-stimulated genes. These findings expand our understanding of STAT3-dependent processes that are key to its function as a pro-viral latency determinant for oncogenic gammaherpesviruses in B cells and may provide novel therapeutic targets.

Enhanced CD19 activity in B cells contributes to immunodeficiency in mice deficient in the ICF syndrome gene Zbtb24.

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a rare autosomal recessive disorder characterized by DNA hypomethylation and antibody deficiency. It is caused by mutations in DNMT3B, ZBTB24, CDCA7, or HELLS. While progress has been made in elucidating the roles of these genes in regulating DNA methylation, little is known about the pathogenesis of the life-threatening hypogammaglobulinemia phenotype. Here, we show that mice deficient in Zbtb24 in the hematopoietic lineage recapitulate the major clinical features of patients with ICF syndrome. Specifically, Vav-Cre-mediated ablation of Zbtb24 does not affect lymphocyte development but results in reduced plasma cells and low levels of IgM, IgG1, and IgA. Zbtb24-deficient mice are hyper and hypo-responsive to T-dependent and T-independent type 2 antigens, respectively, and marginal zone B-cell activation is impaired. Mechanistically, Zbtb24-deficient B cells show severe loss of DNA methylation in the promoter region of Il5ra (interleukin-5 receptor subunit alpha), and Il5ra derepression leads to elevated CD19 phosphorylation. Heterozygous disruption of Cd19 can revert the hypogammaglobulinemia phenotype of Zbtb24-deficient mice. Our results suggest the potential role of enhanced CD19 activity in immunodeficiency in ICF syndrome.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

Uracil-DNA Glycosylase of Murine Gammaherpesvirus 68 Binds Cognate Viral Replication Factors Independently of its Catalytic Residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian Uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect, unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprised of the cognate viral DNA polymerase, vPOL encoded by ORF9 , and the viral DNA polymerase processivity factor, vPPF encoded by ORF59 . MHV68 vUNG colocalized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone, or in combination. Last, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE: Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo . In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus to form a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus associated cancers.

Divergent structures of Mammalian and gammaherpesvirus uracil DNA glycosylases confer distinct DNA binding and substrate activity.

Uracil DNA glycosylase (UNG) removes mutagenic uracil base from DNA to initiate base excision repair (BER). The result is an abasic site (AP site) that is further processed by the high-fidelity BER pathway to complete repair and maintain genome integrity. The gammaherpesviruses (GHVs), human Kaposi sarcoma herpesvirus (KSHV), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68) encode functional UNGs that have a role in viral genome replication. Mammalian and GHVs UNG share overall structure and sequence similarity except for a divergent amino-terminal domain and a leucine loop motif in the DNA binding domain that varies in sequence and length. To determine if divergent domains contribute to functional differences between GHV and mammalian UNGs, we analyzed their roles in DNA interaction and catalysis. By utilizing chimeric UNGs with swapped domains we found that the leucine loop in GHV, but not mammalian UNGs facilitates interaction with AP sites and that the amino-terminal domain modulates this interaction. We also found that the leucine loop structure contributes to differential UDGase activity on uracil in single- versus double-stranded DNA. Taken together we demonstrate that the GHV UNGs evolved divergent domains from their mammalian counterparts that contribute to differential biochemical properties from their mammalian counterparts.

The Longitudinal Analysis of Convergent Antibody VDJ Regions in SARS-CoV-2-Positive Patients Using RNA-Seq.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) is an ongoing pandemic that continues to evolve and reinfect individuals. To understand the convergent antibody responses that evolved over the course of the pandemic, we evaluated the immunoglobulin repertoire of individuals infected by different SARS-CoV-2 variants for similarity between patients. We utilized four public RNA-seq data sets collected between March 2020 and March 2022 from the Gene Expression Omnibus (GEO) in our longitudinal analysis. This covered individuals infected with Alpha and Omicron variants. In total, from 269 SARS-CoV-2-positive patients and 26 negative patients, 629,133 immunoglobulin heavy-chain variable region V(D)J sequences were reconstructed from sequencing data. We grouped samples based on the SARS-CoV-2 variant type and/or the time they were collected from patients. Our comparison of patients within each SARS-CoV-2-positive group found 1011 common V(D)Js (same V gene, J gene and CDR3 amino acid sequence) shared by more than one patient and no common V(D)Js in the noninfected group. Taking convergence into account, we clustered based on similar CDR3 sequence and identified 129 convergent clusters from the SARS-CoV-2-positive groups. Within the top 15 clusters, 4 contain known anti-SARS-CoV-2 immunoglobulin sequences with 1 cluster confirmed to cross-neutralize variants from Alpha to Omicron. In our analysis of longitudinal groups that include Alpha and Omicron variants, we find that 2.7% of the common CDR3s found within groups were also present in more than one group. Our analysis reveals common and convergent antibodies, which include anti-SARS-CoV-2 antibodies, in patient groups over various stages of the pandemic.

A Recombinant Antibody For Tracking Murine Gammaherpesvirus 68 Uracil DNA Glycosylase Expression.

Antibodies are powerful tools to detect expressed proteins. However off-target recognition can confound their use. Therefore, careful characterization is needed to validate specificity in distinct applications. Here we report the sequence and characterization of a mouse recombinant antibody that specifically detects ORF46 of murine gammaherpesvirus 68 (MHV68). This ORF encodes the viral uracil DNA glycosylase (vUNG). The antibody does not recognize murine uracil DNA glycosylase and is useful in detecting vUNG expressed in virally infected cells. It can detect expressed vUNG in cells via immunostaining and microscopy or flow cytometry analysis. The antibody can detect vUNG from lysates of expressing cells via immunoblot under native conditions but not denaturing conditions. This suggests it recognizes a confirmational based epitope. Altogether this manuscript describes the utility of the anti-vUNG antibody and suitability for use in studies of MHV68 infected cells.

Characterization of a mouse model of ICF syndrome reveals enhanced CD19 activation in inducing hypogammaglobulinemia.

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is a rare autosomal recessive disorder characterized by DNA hypomethylation and antibody deficiency. It is caused by mutations in DNMT3B, ZBTB24, CDCA7 or HELLS . While progress has been made in elucidating the roles of these genes in regulating DNA methylation, little is known about the pathogenesis of the life-threatening hypogammaglobulinemia phenotype. Here we show that mice deficient for Zbtb24 in the hematopoietic lineage recapitulate major clinical features of patients with ICF syndrome. Specifically, Vav-Cre-mediated ablation of Zbtb24 does not affect lymphocyte development but results in reduced plasma cells and low levels of IgM, IgG1 and IgA. Zbtb24 -deficient mice are hyper- and hypo-responsive to T-dependent and Tindependent type 2 antigens, respectively, and marginal zone B cell activation is impaired. B cells from Zbtb24 -deficient mice display elevated CD19 phosphorylation. Heterozygous disruption of Cd19 can revert the hypogammaglobulinemia phenotype in these mice. Mechanistically, Il5ra (interleukin-5 receptor subunit alpha) is derepressed in Zbtb24 -deficient B cells, and elevated IL-5 signaling enhances CD19 phosphorylation. Our results reveal a novel link between IL-5 signaling and CD19 activation and suggest that abnormal CD19 activity contributes to immunodeficiency in ICF syndrome. SIGNIFICANCE STATEMENT: ICF syndrome is a rare immunodeficiency disorder first reported in the 1970s. The lack of appropriate animal models has hindered the investigation of the pathogenesis of antibody deficiency, the major cause of death in ICF syndrome. Here we show that, in mice, disruption of Zbtb24 , one of the ICF-related genes, in the hematopoietic lineage results in low levels of immunoglobulins. Characterization of these mice reveals abnormal B cell activation due to elevated CD19 phosphorylation. Mechanistically, Il5ra (interleukin-5 receptor subunit alpha) is derepressed in Zbtb24 -deficient B cells, and increased IL-5 signaling enhances CD19 phosphorylation.

B cell-intrinsic STAT3-mediated support of latency and interferon suppression during murine gammaherpesvirus 68 infection revealed through an in vivo competition model.

Cancers associated with the oncogenic gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, are notable for their constitutive activation of the transcription factor STAT3. To better understand the role of STAT3 during gammaherpesvirus latency and immune control, we utilized murine gammaherpesvirus 68 (MHV68) infection. Genetic deletion of STAT3 in B cells of CD19cre/+Stat3f/f mice reduced peak latency approximately 7-fold. However, infected CD19cre/+Stat3f/f mice exhibited disordered germinal centers and heightened virus-specific CD8 T cell responses compared to WT littermates. To circumvent the systemic immune alterations observed in the B cell-STAT3 knockout mice and more directly evaluate intrinsic roles for STAT3, we generated mixed bone marrow chimeras consisting of WT and STAT3-knockout B cells. Using a competitive model of infection, we discovered a dramatic reduction in latency in STAT3-knockout B cells compared to their WT B cell counterparts in the same lymphoid organ. RNA sequencing of sorted germinal center B cells revealed that STAT3 promotes proliferation and B cell processes of the germinal center but does not directly regulate viral gene expression. Last, this analysis uncovered a STAT3-dependent role for dampening type I IFN responses in newly infected B cells. Together, our data provide mechanistic insight into the role of STAT3 as a latency determinant in B cells for oncogenic gammaherpesviruses.

Targeting MYC: From understanding its biology to drug discovery.

The MYC oncogene is considered to be a high priority target for clinical intervention in cancer patients due to its aberrant activation in more than 50% of human cancers. Direct small molecule inhibition of MYC has traditionally been hampered by its intrinsically disordered nature and lack of both binding site and enzymatic activity. In recent years, however, a number of strategies for indirectly targeting MYC have emerged, guided by the advent of protein structural information and the growing set of computational tools that can be used to accelerate the hit to lead process in medicinal chemistry. In this review, we provide an overview of small molecules developed for clinical applications of these strategies, which include stabilization of the MYC guanine quadruplex, inhibition of BET factor BRD4, and disruption of the MYC:MAX heterodimer. The recent identification of novel targets for indirect MYC inhibition at the protein level is also discussed.

Transcriptional Activation of MYC-Induced Genes by GCN5 Promotes B-cell Lymphomagenesis.

Overexpression of the MYC oncoprotein is an initiating step in the formation of several cancers. MYC frequently recruits chromatin-modifying complexes to DNA to amplify the expression of cancer-promoting genes, including those regulating cell cycle, proliferation, and metabolism, yet the roles of specific modifiers in different cancer types are not well defined. Here, we show that GCN5 is an essential coactivator of cell-cycle gene expression driven by MYC overexpression and that deletion of Gcn5 delays or abrogates tumorigenesis in the Eµ-Myc mouse model of B-cell lymphoma. Our results demonstrate that Gcn5 loss impacts both expression and downstream functions of Myc. SIGNIFICANCE: Our results provide important proof of principle for Gcn5 functions in formation and progression of Myc-driven cancers, suggesting that GCN5 may be a viable target for development of new cancer therapies.

Defining the mutation signatures of DNA polymerase θ in cancer genomes.

DNA polymerase theta (POLQ)-mediated end joining (TMEJ) is a distinct pathway for mediating DNA double-strand break (DSB) repair. TMEJ is required for the viability of BRCA-mutated cancer cells. It is crucial to identify tumors that rely on POLQ activity for DSB repair, because such tumors are defective in other DSB repair pathways and have predicted sensitivity to POLQ inhibition and to cancer therapies that produce DSBs. We define here the POLQ-associated mutation signatures in human cancers, characterized by short insertions and deletions in a specific range of microhomologies. By analyzing 82 COSMIC (Catalogue of Somatic Mutations in Cancer) signatures, we found that BRCA-mutated cancers with a higher level of POLQ expression have a greatly enhanced representation of the small insertion and deletion signature 6, as well as single base substitution signature 3. Using human cancer cells with disruptions of POLQ, we further show that TMEJ dominates end joining of two separated DSBs (distal EJ). Templated insertions with microhomology are enriched in POLQ-dependent distal EJ. The use of this signature analysis will aid in identifying tumors relying on POLQ activity.

Dangerous Liaisons: Gammaherpesvirus Subversion of the Immunoglobulin Repertoire.

A common biologic property of the gammaherpesviruses Epstein-Barr Virus and Kaposi sarcoma herpesvirus is their use of B lymphocytes as a reservoir of latency in healthy individuals that can undergo oncogenic transformation later in life. Gammaherpesviruses (GHVs) employ an impressive arsenal of proteins and non-coding RNAs to reprogram lymphocytes for proliferative expansion. Within lymphoid tissues, the germinal center (GC) reaction is a hub of B cell proliferation and death. The goal of a GC is to generate and then select for a pool of immunoglobulin (Ig) genes that will provide a protective humoral adaptive immune response. B cells infected with GHVs are detected in GCs and bear the hallmark signatures of the mutagenic processes of somatic hypermutation and isotype class switching of the Ig genes. However, data also supports extrafollicular B cells as a reservoir engaged by GHVs. Next-generation sequencing technologies provide unprecedented detail of the Ig sequence that informs the natural history of infection at the single cell level. Here, we review recent reports from human and murine GHV systems that identify striking differences in the immunoglobulin repertoire of infected B cells compared to their uninfected counterparts. Implications for virus biology, GHV-associated cancers, and host immune dysfunction will be discussed.

Gammaherpesvirus-infected germinal center cells express a distinct immunoglobulin repertoire.

The gammaherpesviruses (γHVs), human Kaposi sarcoma-associated herpesvirus (KSHV), EBV, and murine γHV68 are prevalent infections associated with lymphocyte pathologies. After primary infection, EBV and γHV68 undergo latent expansion in germinal center (GC) B cells and persists in memory cells. The GC reaction evolves and selects antigen-specific B cells for memory development but whether γHV passively transients or manipulates this process in vivo is unknown. Using the γHV68 infection model, we analyzed the Ig repertoire of infected and uninfected GC cells from individual mice. We found that infected cells displayed the hallmarks of affinity maturation, hypermutation, and isotype switching but underwent clonal expansion. Strikingly, infected cells displayed distinct repertoire, not found in uninfected cells, with recurrent utilization of certain Ig heavy V segments including Ighv10-1 In a manner observed with KSHV, γHV68 infected cells also displayed lambda light chain bias. Thus, γHV68 subverts GC selection to expand in a specific B cell subset during the process that develops long-lived immunologic memory.

B cells and tertiary lymphoid structures promote immunotherapy response.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.