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PurposeThe COVID PROFILE cohort is a longitudinal clinical study based in Victoria Australia, which was established to understand immunity to SARS-CoV-2 in alow transmission population settingand to identify immunological markers of long-term immunity and immune-dysregulation after both infection and vaccination. Additionally, this cohort was established as a biobank resource for researchers to address other health-related immunological questions.ParticipantsWe enrolled 178 adult community members, including household contacts, who had either recovered from a SARS-CoV-2 infection or were SARS-CoV-2 naïve. Only participants ≥18 years of age and, in the case of female participants, non-pregnant women at the time of enrollment were included in the study. Detailed COVID-19 clinical data, vaccination status, medical history and demographics was collected.Findings to dateAt enrollment, we found that 87.8% of COVID-19 recovered individuals were seropositive with detectable levels of anti-SARS-CoV-2 IgG antibodies. Seronegative COVID-19 recovered individuals included asymptomatic individuals or participants that were enrolled more than 12 months after their COVID-19 diagnosis. Except for one individual who was seropositive at baseline despite a previous SARS-CoV-2 PCR negative diagnosis, all household contacts and other community members enrolled as SARS-CoV-2 PCR negative, were seronegative for all SARS-CoV-2-specific antibodies tested. The infection rate (re-infection or new infection) during 24 months of the study was 42.7%, as determined by either rapid antigen tests, PCRs or serology screens. Of the SARS-CoV-2 recovered participants, 32.6% reported ongoing symptoms at enrollment of which 47% had already experienced ongoing symptoms for more than 12 weeks.Future PlansCOVID PROFILE will be used to comprehensively understand temporal immunity to SARS-CoV-2 and COVID-19 vaccines and to understand the impact of host immunological composition on such immunity and symptom severity. Additionally, studies focusing on understanding immunity following breakthrough infections and immunological risk factors that contribute towards development of long COVID are planned.Limitations/Strengths of the studyExtensive clinical information is available and longitudinal samples (blood, saliva and oropharyngeal swabs) collected at regular intervals up to 2.5 years after initial enrolment.This low SARS-CoV-2 transmission population cohort, enables exploration of difference in both the acquisition and maintenance of naturally acquired and vaccine-induced immunity not confounded by prior, frequent and/or undetected exposures.Extensive biobank of numerous blood fractions and biospecimen enables further exploration of mucosal immunity, nasal microbiome and humoral and cellular immunity over time.The cohort may be limited in addressing research questions regarding outcomes and risk factors and their associations where low incidence is expected.

SUMMARYThe transcription factor Myc is critically important in driving cell proliferation, a function that is frequently dysregulated in cancer. To avoid this dysregulation Myc is tightly controlled by numerous layers of regulation. One such layer is the use of distal regulatory enhancers to drive Myc expression. Here, using chromosome conformation capture to examine B cells of the immune system in the first hours after their activation, we reveal a previously unidentified enhancer of myc. The interactivity of this enhancer coincides with a dramatic, but discrete, spike in Myc expression 3 hours post-activation. However, genetic deletion of this region, has little impact on Myc expression, Myc protein level or in vitro and in vivo cell proliferation. Examination of the enhancer deleted regulatory landscape suggests that enhancer redundancy likely sustains Myc expression. This work highlights not only the importance of temporally examining enhancers, but also the complexity and dynamics of the regulation of critical genes such as Myc.

During viral infection, immune cells coordinate the induction of inflammatory responses that clear infection and humoral responses that promote protection. CD4+T-cell differentiation sits at the center of this axis. Differentiation toward T-helper 1 (Th1) cells mediates inflammation and pathogen clearance, while T follicular helper (Tfh) cells facilitate germinal center (GC) reactions for the generation of high-affinity antibodies and immune memory. While Th1 and Tfh differentiation occurs in parallel, these CD4+T-cell identities are mutually exclusive, and progression toward these ends is determined via the upregulation of T-bet and Bcl6, respectively. These lineage-defining transcription factors act in concert with multiple networks of transcriptional regulators that tip the T-bet and Bcl6 axis in CD4+T-cell progenitors to either a Th1 or Tfh fate. It is now clear that these transcriptional networks are guided by cytokine cues that are not only varied between distinct viral infections but also dynamically altered throughout the duration of infection. Thus, multiple intrinsic and extrinsic factors combine to specify the fate, plasticity, and function of Th1 and Tfh cells during infection. Here, we review the current information on the mode of action of the lineage-defining transcription factors Bcl6 and T-bet and how they act individually and in complex to govern CD4+T-cell ontogeny. Furthermore, we outline the multifaceted transcriptional regulatory networks that act upstream and downstream of Bcl6 and T-bet to tip the differentiation equilibrium toward either a Tfh or Th1 fate and how these are impacted by dynamic inflammatory cues.

The chemokine receptor CXCR3 is expressed on immune cells to co-ordinate lymphocyte activation and migration. CXCR3 binds three chemokine ligands, CXCL9, CXCL10 and CXCL11. These ligands display distinct expression patterns and ligand signaling biases; however, how each ligand functions individually and collaboratively is incompletely understood. CXCL9 and CXCL10 are considered pro-inflammatory chemokines during viral infection, while CXCL11 may induce a tolerizing state. The investigation of the individual role of CXCL11 in vivo has been hampered as C57BL/6 mice carry several mutations that result in a null allele. Here, CRISPR/Cas9 was used to correct these mutations on a C57BL/6 background. It was validated that CXCL11

Recent studies have established that memory B cells, largely thought to be circulatory in the blood, can take up long-term residency in inflamed tissues, analogous to widely described tissue-resident T cells. The dynamics of recruitment and retention of memory B cells to tissues and their immunological purpose remains unclear. Here, we characterized tissue-resident memory B cells (B RM ) that are stably maintained in the lungs of mice after pulmonary influenza infection. Influenza - specific B RM were localized within inducible bronchus-associated lymphoid tissues (iBALTs) and displayed transcriptional signatures distinct from classical memory B cells in the blood or spleen while showing partial overlap with memory B cells in lung-draining lymph nodes. We identified lung-resident markers, including elevated expression of CXCR3, CCR6, and CD69, on hemagglutinin (HA)– and nucleoprotein (NP)–specific lung B RM . We found that CCR6 facilitates increased recruitment and/or retention of B RM in lungs and differentiation into antibody-secreting cells upon recall. Although expression of CXCR3 and CCR6 was comparable in total and influenza-specific memory B cells isolated across tissues of human donors, CD69 expression was higher in memory B cells from lung and draining lymph nodes of human organ donors relative to splenic and PBMC-derived populations, indicating that mechanisms underpinning B RM localization may be evolutionarily conserved. Last, we demonstrate that human memory B cells in lungs are transcriptionally distinct to populations in lung-draining lymph nodes or PBMCs. These data suggest that B RM may constitute a discrete component of B cell immunity, positioned at the lung mucosa for rapid humoral response against respiratory viral infections.

The spleen is a compartmentalized organ that serves as a blood filter and safeguard of systemic immune surveillance. Labyrinthine networks of fibroblastic stromal cells construct complex niches within the white pulp and red pulp that are important for tissue homeostasis and immune activation. However, the identity and roles of the global splenic fibroblastic stromal cells in homeostasis and immune responses are poorly defined. Here, we performed a cellular and molecular dissection of the splenic reticular stromal cell landscape. We found that white pulp fibroblastic reticular cells (FRCs) responded robustly during acute viral infection, but this program of gene regulation was suppressed during persistent viral infection. Single-cell transcriptomic analyses in mice revealed diverse fibroblast cell niches and unexpected heterogeneity among podoplanin-expressing cells that include glial, mesothelial, and adventitial cells in addition to FRCs. We found analogous fibroblastic stromal cell diversity in the human spleen. In addition, we identify the transcription factor SpiB as a critical regulator required to support white pulp FRC differentiation, homeostatic chemokine expression, and antiviral T cell responses. Together, our study provides a comprehensive map of fibroblastic stromal cell types in the spleen and defines roles for red and white pulp fibroblasts for splenic function and orchestration of immune responses.

High-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, includingMybl1,Myof,Gas7andAtoh8. Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.

The lymph node plays a critical role in mounting an adaptive immune response to infection, clearance of foreign pathogens, and cancer immunosurveillance. Within this complex structure, intranodal migration is vital for CD8

T follicular helper (Tfh) cells are an important component of the germinal centre (GC)-mediated humoral immunity. Yet, how regulation of Tfh- GC responses impacts on effective responses to helminth infection are poorly understood. Here we show that chronic helminth Trichuris muris infection fails to induce Tfh-GC B cell responses, with Tfh cells expressing T-bet and IFN-γ. In contrast, Tfh cells that express GATA-3 and IL-4 dominate responses to an acute, resolving infection. Accordingly, heightened expression and increased chromatin accessibility of Th1- and Th2 cell-associated genes is observed in chronic and acute induced Tfh cells, respectively. However, both acute and chronic Tfh cell populations retained the capacity to produce IL-21 in spite of the Th-biased response. Blockade of Tfh-GC interactions impaired type 2 immunity, highlighting the protective role of GC-dependent Th2-like Tfh cell responses against helminths. Collectively, these results provide new insights into the protective roles of Tfh-GC responses and identify distinct transcriptional and epigenetic features of Tfh cells that emerge during resolving or chronic helminth infections.Author summaryAbout a quarter of the world population is afflicted with parasitic worm infection. Although deworming drugs can reduce the levels of the infection, they fail to prevent reinfections. Therefore, the most sustainable goal is to develop vaccines against human helminth parasites, which has been extremely challenging due to the lack of understanding of host-parasite interactions. While the protective roles of T helper 2 (Th2) cells are well established, the regulation of T follicular helper (Tfh) cells and their roles during helminth infection remain poorly defined. In this study, we describe the differential regulation of Tfh cell responses during chronic non-protective vs acute protective responses during helminth infection. We show that Tfh cells during chronic infection are rare and have strikingly different characteristics to acute-induced Tfh cells, which appear to be more like Th2 cells. Specifically, we show that blockade of Th2-like Tfh cell response during acute infection results in the host failing to expel the worms. Our study identifies that Tfh cell populations that emerge during chronic and acute infection are strikingly heterogeneous and critically important in mediating protective immune responses against helminths.

NK cells are cytotoxic lymphocytes with a key role in limiting tumour metastases. In mice, the NK cell lineage continually expresses high levels of the Inhibitor of DNA-binding 2 (Id2) protein and loss of Id2 is incongruous with their survival due to aberrant E-protein target gene activity. Using novel Id2 and E-protein antibodies that detect both mouse and human proteins, we have extensively characterised Id2 and E-protein expression in murine and human NK cells. We detected clear expression of E2 A and HEB, and to a lesser extent E2-2 in murine NK cells. In contrast HEB appears to be the major E-protein expressed in human NK cells, with minor E2-2 expression and surprisingly, no E2 A detected in primary NK cells nor human NK cell lines. These novel antibodies are also functional in immunofluorescence and immunoprecipitation. Mass spectrometry analysis of Id2 immuno-precipitated from murine NK cells revealed a number of novel associated proteins including several members of the SWI/SNF-related matrix-associated actin-dependent regulator chromatin (SMARC) and Mediator complex (MED) families. Taken together, these data highlight the utility of novel Id2 and E-protein antibodies and caution against mouse models for understanding Id2/E-protein biology in NK cells given their clearly disparate expression patternbetween species.

Summary: Despite the key role that antibodies play in protection, the cellular processes mediating the acquisition of humoral immunity against malaria are not fully understood. Using an infection model of severe malaria, we find that germinal center (GC) B cells upregulate the transcription factor T-bet during infection. Molecular and cellular analyses reveal that T-bet in B cells is required not only for IgG2c switching but also favors commitment of B cells to the dark zone of the GC. T-bet was found to regulate the expression of Rgs13 and CXCR3, both of which contribute to the impaired GC polarization observed in the absence of T-bet, resulting in reduced IghV gene mutations and lower antibody avidity. These results demonstrate that T-bet modulates GC dynamics, thereby promoting the differentiation of B cells with increased affinity for antigen. : Antibody responses play a pivotal role in clinical immunity to malaria. Ly et al. report that that germinal center (GC) B cells upregulate the transcription factor T-bet during infection. T-bet favors commitment of B cells to the GC dark zone, thereby augmenting immunoglobulin gene mutation rates and antibody affinity maturation. Keywords: malaria, T-bet, B cells, germinal center, antibodies, affinity maturation

To optimize immunity to pathogens, B lymphocytes generate plasma cells with functionally diverse antibody isotypes. By lineage tracing single cells within differentiating B cell clones, we identified the heritability of discrete fate controlling mechanisms to inform a general mathematical model of B cell fate regulation. Founder cells highly influenced clonal plasma-cell fate, whereas class switch recombination (CSR) was variegated within clones. In turn, these CSR patterns resulted from independent all-or-none expression of both activation-induced cytidine deaminase (AID) and IgH germline transcription (GLT), with the latter being randomly re-expressed after each cell division. A stochastic model premised on these molecular transition rules accurately predicted antibody switching outcomes under varied conditions in vitro and during an immune response in vivo. Thus, the generation of functionally diverse antibody types follows rules of autonomous cellular programming that can be adapted and modeled for the rational control of antibody classes for potential therapeutic benefit.

Summary: Correct positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen-presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induces tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters are “hotspots” for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolongs T-APC interactions and boosts function. Both the frequency and range of these clusters are enhanced via a T helper 1 (Th1)-intrinsic, interferon-gamma (IFNγ)-dependent positive-feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

International audience; Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

The relationship between the extrinsic environment and the internal transcriptional network is circular. Naive T cells first engage with antigen‐presenting cells to set transcriptional differentiation networks in motion. In turn, this regulates specific chemokine receptors that direct migration into distinct lymph node niches. Movement into these regions brings newly activated T cells into contact with accessory cells and cytokines that reinforce the differentiation programming to specify T cell function. We and others have observed similarities in the transcriptional networks that specify both CD4+ T follicular helper (TFH) cells and CD8+ central memory stem‐like (TSCM) cells. Here, we compare and contrast the current knowledge for these shared differentiation programs, compared to their effector counterparts, CD4+ T‐helper 1 (TH1) and CD8+ short‐lived effector (TSLEC) cells. Understanding the interplay between cellular interactions and transcriptional programming is essential to harness T cell differentiation that is fit for purpose; to stimulate potent T cell effector function for the elimination of chronic infection and cancer; or to amplify the formation of humoral immunity and longevity of cellular memory to prevent infectious diseases.

SUMMARYCorrect positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induced tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters were ‘hot spots’ for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolonged T-APC interactions and boosted function. Both the frequency and range of these clusters were enhanced via a Th1-intrinsic, IFNγ-dependent positive feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

Correct positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen-presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induces tissue-wide de novo generation of discrete perivascular CXCL10

Summary: Histone modifiers are essential for the ability of immune cells to reprogram their gene expression during differentiation. The recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like) induces oncogenic gene expression in a subset of B cell leukemias. Despite its importance, its role in the humoral immune system is unclear. Here, we demonstrate that DOT1L is a critical regulator of B cell biology. B cell development is defective in Dot1lf/fMb1Cre/+ mice, culminating in a reduction of peripheral mature B cells. Upon immunization or influenza infection of Dot1lf/fCd23Cre/+ mice, class-switched antibody-secreting cells are significantly attenuated and germinal centers fail to form. Consequently, DOT1L is essential for B cell memory formation. Transcriptome, pathway, and histological analyses identified a role for DOT1L in reprogramming gene expression for appropriate localization of B cells during the initial stage of the response. Together, these results demonstrate an essential role for DOT1L in generating an effective humoral immune response.

Plasmacytoid dendritic cells (pDCs) play a critical role in bridging the innate and adaptive immune systems. pDCs are specialized type I interferon (IFN) producers, which has implicated them as initiators of autoimmune pathogenesis. However, little is known about the downstream effectors of type I IFN signaling that amplify autoimmune responses. Here, we have used a chemokine reporter mouse to determine the CXCR3 ligand responses in DCs subsets. Following TLR7 stimulation, conventional type 1 and type 2 DCs (cDC1 and cDC2, respectively) uniformly upregulate CXCL10. By contrast, the proportion of chemokine positive pDCs was significantly less, and stable CXCL10+ and CXCL10- populations could be distinguished. CXCL9 expression was induced in all cDC1s, in half of the cDC2 but not by pDCs. The requirement for IFNAR signaling for chemokine reporter expression was interrogated by receptor blocking and deficiency and shown to be critical for CXCR3 ligand expression in Flt3-ligand-derived DCs. Chemokine-producing potential was not concordant with the previously identified markers of pDC heterogeneity. Finally, we show that CXCL10+ and CXCL10- populations are transcriptionally distinct, expressing unique transcriptional regulators, IFN signaling molecules, chemokines, cytokines, and cell surface markers. This work highlights CXCL10 as a downstream effector of type I IFN signaling and suggests a division of labor in pDCs subtypes that likely impacts their function as effectors of viral responses and as drivers of inflammation.

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

Infection or inflammation of the skin recruits effector CD8+ T cells that enter the epidermis and form populations of long-lived tissue-resident memory T (TRM) cells. These skin TRM cells migrate within the constrained epidermal environment by extending multiple dynamic dendritic projections and squeezing between keratinocytes to survey the tissue for pathogens. In this study, we examined the signals required for this distinctive mode of T cell migration by inhibiting key cytoskeletal components and performing intravital two-photon microscopy to visualize TRM cell behavior. We found that TRM cell motility and dendrite formation required an intact actomyosin cytoskeleton and the Rho-associated coiled-coil containing kinases. We also identified an essential role for microtubules for maintaining skin TRM cell shape and cellular integrity. We reveal a role for pertussis toxin–sensitive signaling for TRM cell dendritic morphology and migration that is independent of CXCR3 or CXCR6, or the skin-selective chemokine receptors CCR10 and CCR8. However, we found that CXCR6 and CCR10 expression by CD8+ T cells was required for the optimal formation of memory T cell populations, in particular TRM cell populations in the skin.

The lymphoid tissue that drains the upper respiratory tract represents an important induction site for cytotoxic T lymphocyte (CTL) immunity to airborne pathogens and intranasal vaccines. Here, we investigated the role of the nasal-associated lymphoid tissues (NALTs), which are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage, in the initial priming and recall expansion of CD8+ T cells following an upper respiratory tract infection with a pathogenic influenza virus and immunization with a live attenuated influenza virus vaccine. Whereas NALTs served as the induction site for the recall expansion of memory CD8+ T cells following influenza virus infection or vaccination, they failed to support activation of naive CD8+ T cells. Strikingly, NALTs, unlike other lymphoid tissues, were not routinely surveyed during the steady state by circulating T cells. The selective recruitment of memory T cells into these lymphoid structures occurred in response to infection-induced elevation of the chemokine CXCL10, which attracted CXCR3+ memory CD8+ T cells. These results have significant implications for intranasal vaccines, which deliver antigen to mucosal-associated lymphoid tissue and aim to elicit protective CTL-mediated immunity.

The combination of single-cell RNA-seq and in vivo CRISPR–Cas9 screens reveal a new circuit that directs germinal center B cells toward a memory B cell phenotype during viral infection.

T cells dynamically interact with multiple, distinct cellular subsets to determine effector and memory differentiation. Here, we developed a platform to quantify cell location in three dimensions to determine the spatial requirements that direct T cell fate. After viral infection, we demonstrated that CD8

Protein-based, self-assembling nanoparticles elicit superior immunity compared with soluble protein vaccines, but the immune mechanisms underpinning this effect remain poorly defined. Here, we investigated the immunogenicity of a prototypic ferritin-based nanoparticle displaying influenza hemagglutinin (HA) in mice and macaques. Vaccination of mice with HA-ferritin nanoparticles elicited higher serum antibody titers and greater protection against experimental influenza challenge compared with soluble HA protein. Germinal centers in the draining lymph nodes were expanded and persistent following HA-ferritin vaccination, with greater deposition of antigen that colocalized with follicular dendritic cells. Our findings suggest that a highly ordered and repetitive antigen array may directly drive germinal centers through a B cell-intrinsic mechanism that does not rely on ferritin-specific T follicular helper cells. In contrast to mice, enhanced immunogenicity of HA-ferritin was not observed in pigtail macaques, where antibody titers and lymph node immunity were comparable to soluble vaccination. An improved understanding of factors that drive nanoparticle vaccine immunogenicity in small and large animal models will facilitate the clinical development of nanoparticle vaccines for broad and durable protection against diverse pathogens.

Phenotypically diverse memory CD8(+) T cells are present in the lungs that either re-circulate or reside within the tissue. Understanding the key cellular interactions that regulate the generation and then persistence of these different subsets is of great interest. Recently, DNGR-1(+) dendritic cell (DC) mediated priming was reported to control the generation of lung resident but not circulating memory cells following respiratory viral infection. Here, we report an important role for Ly6C(+) inflammatory monocytes (IMs) in contributing to the persistence of memory CD8(+) T cells but not their generation. Effector CD8(+) T cells expanded and contracted normally in the absence of IMs, but the memory compartment declined significantly over time. Quite unexpectedly, this defect was confined to tissue resident and circulating CXCR3(hi)CX3CR1(lo) memory cells but not CXCR3(hi)CX3CR1(int) and CXCR3(lo)CX3CR1(hi) subsets. Thus, two developmentally distinct innate cells orchestrate the generation and persistence of memory T cell subsets following a respiratory virus infection.

SUMMARYHistone modifiers are essential molecular regulators that underpin the ability of immune cells to reprogram their gene expression during differentiation. The recruitment of the histone methyltransferase DOT1L induces oncogenic gene expression in a subset of B cell leukemia. Despite its importance, little is known about its role in the humoral immune system. Herein, we demonstrate that DOT1L is a critical regulator of B cell biology. Dot1lf/fMb1Cre/+ mice had a block in B cell development, culminating in a significant reduction of mature B cells in the periphery. Upon immunization or influenza infection of Dot1lf/fCd23Cre/+ mice, germinal centers failed to form and class-switched antibody-secreting cells were significantly attenuated. Consequently, immunized mice revealed that DOT1L was essential for the formation of B cell memory populations. Transcriptome, pathway and histological analysis identified a key role for DOT1L in reprogramming gene expression for migration and localization during the initial stages of a humoral response. Together, these results demonstrate an essential role for DOT1L in antigen-dependent B cell differentiation and hence, in generating an effective and lasting humoral immune response.