Distinct baseline immune characteristics associated with responses to conjugated and unconjugated pneumococcal polysaccharide vaccines in older adults.

Pneumococcal infections cause serious illness and death among older adults. The capsular polysaccharide vaccine PPSV23 and conjugated alternative PCV13 can prevent these infections; yet, underlying immunological responses and baseline predictors remain unknown. We vaccinated 39 older adults (>60 years) with PPSV23 or PCV13 and observed comparable antibody responses (day 28) and plasmablast transcriptional responses (day 10); however, the baseline predictors were distinct. Analyses of baseline flow cytometry and bulk and single-cell RNA-sequencing data revealed a baseline phenotype specifically associated with weaker PCV13 responses, which was characterized by increased expression of cytotoxicity-associated genes, increased frequencies of CD16+ natural killer cells and interleukin-17-producing helper T cells and a decreased frequency of type 1 helper T cells. Men displayed this phenotype more robustly and mounted weaker PCV13 responses than women. Baseline expression levels of a distinct gene set predicted PPSV23 responses. This pneumococcal precision vaccinology study in older adults uncovered distinct baseline predictors that might transform vaccination strategies and initiate novel interventions.

Natural killer cell memory.

Natural killer (NK) cells are bone marrow­derived granular lymphocytes that have a key role in immune defense against viral and bacterial infections and malignancies. NK cells are traditionally defined as cells of the innate immune response because they lack RAG recombinase­dependent clonal antigen receptors. However, evidence suggests that specific subsets of mouse NK cells can nevertheless develop long-lived and highly specific memory to a variety of antigens. Here we review published evidence of NK cell­mediated, RAG-independent adaptive immunity. We also compare and contrast candidate mechanisms for mammalian NK cell memory and antigen recognition with other examples of RAG-independent pathways that generate antigen receptor diversity in non-mammalian species and discuss NK cell memory in the context of lymphocyte evolution.

Matrix Protein 2 Extracellular Domain-Specific Monoclonal Antibodies Are an Effective and Potentially Universal Treatment for Influenza A.

Influenza virus infection causes significant morbidity and mortality worldwide. Humans fail to make a universally protective memory immune response to influenza A. Hemagglutinin and Neuraminidase undergo antigenic drift and shift, resulting in new influenza A strains to which humans are naive. Seasonal vaccines are often ineffective and escape mutants have been reported to all treatments for influenza A. In the absence of a universal influenza A vaccine or treatment, influenza A will remain a significant threat to human health. The extracellular domain of the M2-ion channel (M2e) is an ideal antigenic target for a universal therapeutic agent, as it is highly conserved across influenza A serotypes, has a low mutation rate, and is essential for viral entry and replication. Previous M2e-specific monoclonal antibodies (M2e-MAbs) show protective potential against influenza A, however, they are either strain specific or have limited efficacy. We generated seven murine M2e-MAbs and utilized in vitro and in vivo assays to validate the specificity of our novel M2e-MAbs and to explore the universality of their protective potential. Our data shows our M2e-MAbs bind to M2e peptide, HEK cells expressing the M2 channel, as well as, influenza virions and MDCK-ATL cells infected with influenza viruses of multiple serotypes. Our antibodies significantly protect highly influenza A virus susceptible BALB/c mice from lethal challenge with H1N1 A/PR/8/34, pH1N1 A/CA/07/2009, H5N1 A/Vietnam/1203/2004, and H7N9 A/Anhui/1/2013 by improving survival rates and weight loss. Based on these results, at least four of our seven M2e-MAbs show strong potential as universal influenza A treatments.IMPORTANCE Despite a seasonal vaccine and multiple therapeutic treatments, Influenza A remains a significant threat to human health. The biggest obstacle is producing a vaccine or treatment for influenza A is their universality or efficacy against not only seasonal variances in the influenza virus, but also against all human, avian, and swine serotypes and, therefore, potential pandemic strains. M2e has huge potential as a target for a vaccine or treatment against influenza A. It is the most conserved external protein on the virus. Antibodies against M2e have made it to clinical trials, but not succeeded. Here, we describe novel M2e antibodies produced in mice that are not only protective at low doses, but that we extensively test to determine their universality and found to be cross protective against all strains tested. Additionally, our work begins to elucidate the critical role of isotype for an influenza A monoclonal antibody therapeutic.

Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses.

Hepatic natural killer (NK) cells mediate antigen-specific contact hypersensitivity (CHS) in mice deficient in T cells and B cells. We report here that hepatic NK cells, but not splenic or naive NK cells, also developed specific memory of vaccines containing antigens from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus type 1 (HIV-1). Adoptive transfer of virus-sensitized NK cells into naive recipient mice enhanced the survival of the mice after lethal challenge with the sensitizing virus but not after lethal challenge with a different virus. NK cell memory of haptens and viruses depended on CXCR6, a chemokine receptor on hepatic NK cells that was required for the persistence of memory NK cells but not for antigen recognition. Thus, hepatic NK cells can develop adaptive immunity to structurally diverse antigens, an activity that requires NK cell-expressed CXCR6.

Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation.

The skin has a dual function as a barrier and a sensory interface between the body and the environment. To protect against invading pathogens, the skin harbours specialized immune cells, including dermal dendritic cells (DDCs) and interleukin (IL)-17-producing γδ T (γδT17) cells, the aberrant activation of which by IL-23 can provoke psoriasis-like inflammation. The skin is also innervated by a meshwork of peripheral nerves consisting of relatively sparse autonomic and abundant sensory fibres. Interactions between the autonomic nervous system and immune cells in lymphoid organs are known to contribute to systemic immunity, but how peripheral nerves regulate cutaneous immune responses remains unclear. We exposed the skin of mice to imiquimod, which induces IL-23-dependent psoriasis-like inflammation. Here we show that a subset of sensory neurons expressing the ion channels TRPV1 and Nav1.8 is essential to drive this inflammatory response. Imaging of intact skin revealed that a large fraction of DDCs, the principal source of IL-23, is in close contact with these nociceptors. Upon selective pharmacological or genetic ablation of nociceptors, DDCs failed to produce IL-23 in imiquimod-exposed skin. Consequently, the local production of IL-23-dependent inflammatory cytokines by dermal γδT17 cells and the subsequent recruitment of inflammatory cells to the skin were markedly reduced. Intradermal injection of IL-23 bypassed the requirement for nociceptor communication with DDCs and restored the inflammatory response. These findings indicate that TRPV1(+)Nav1.8(+) nociceptors, by interacting with DDCs, regulate the IL-23/IL-17 pathway and control cutaneous immune responses.

AuNP-M2e + sCpG vaccination of juvenile mice generates lifelong protective immunity to influenza A virus infection.

BACKGROUND: Influenza virus infection causes significant morbidity and mortality worldwide. Humans fail to make a universally protective memory response to influenza A because of high mutation rates in the immune-dominant influenza epitopes. We seek the development of a universal influenza A vaccine. The extracellular domain of the M2-ion channel (M2e) is an ideal antigenic target, as it is highly conserved, has a low mutation rate, and is essential for viral entry and replication. Considering the potential of a universal influenza vaccine for lifelong protection, we aimed to examine this potential using a recently published gold nanoparticle M2e vaccine with CpG as an adjuvant (AuNP-M2e + sCpG). Intranasal vaccination induces an M2e-specific memory response, which is protective against lethal infection with H1N1, H3N2, and H5N1 serotypes, in young BALB/c mice. Protection with AuNP-M2e + sCpG has been published up to 8 months after vaccination. However, the highest risk population during most influenza seasons is adults over 65 years old. Additionally, the efficacy of many vaccines decrease after aging and requiring booster vaccinations to remain effective. RESULTS: To determine if the AuNP-M2e + sCpG vaccine is a viable option as a universal vaccination capable of protection through geriatric age, we tested if the AuNP-M2e + sCpG vaccination loses efficacy after aging mice to geriatric age (over 18 months). Our data shows that mice aged 15 months after vaccination (~ 18-21 months old) retain significant M2e-specific antibody titers in total IgG, IgG1, IgG2a, and IgG2b. These mice are significantly protected from lethal influenza challenge (H1N1, 8.3 PFU). Further, these antibody titers increase upon infection with influenza A and remain elevated for 3 months, suggesting the elderly mice retain effective M2e-specific memory B cells. CONCLUSIONS: Our results demonstrate that protective M2e-specific memory in mice developed at a young age can persist until geriatric age. Additionally, this memory is protective and M2e-specific B cells produced by vaccination with AuNP-M2e + sCpG are maintained and functional. If the results of this study persist in humans, they suggest that a universal influenza A vaccine could be administered early in life and maintain lifelong protection into geriatric age.

Redefining Memory: Building the Case for Adaptive NK Cells.

Classically, natural killer (NK) cells have been defined by nonspecific innate killing of virus-infected and tumor cells. However, burgeoning evidence suggests that the functional repertoire of NK cells is far more diverse than has been previously appreciated, thus raising the possibility that there may be unexpected functional specialization and even adaptive capabilities among NK cell subpopulations. Some of the first evidence that NK cells respond in an antigen-specific fashion came from experiments revealing that subpopulations of murine NK cells were able to respond to a specific murine cytomegalovirus (MCMV) protein and that in the absence of T and B cells, murine NK cells also mediated adaptive immune responses to a secondary challenge with specific haptens. These data have been followed by demonstrations of NK cell memory of viruses and viral antigens in mice and primates. Herein, we discuss different forms of NK cell antigen specificity and how these responses may be tuned to specific viral pathogens, and we provide assessment of the current literature that may explain molecular mechanisms of the novel phenomenon of NK cell memory.

Specific combinations of donor and recipient KIR-HLA genotypes predict for large differences in outcome after cord blood transplantation.

The ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1 HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft.

Natural Killer Cells Response to IL-2 Stimulation Is Distinct between Ascites with the Presence or Absence of Malignant Cells in Ovarian Cancer Patients.

Peritoneal ascites are a distinguishable feature of patients with advanced epithelial ovarian cancer (EOC). The presence of different lymphocyte subsets has been reported in EOC-associated ascites, which also can or not contain malignant cells. The goal of this study was to analyze the functional characteristics of natural killer (NK) cells from EOC-associated ascites in terms of their expression of activating receptors and ascites' contents of lymphocyte subtypes, cytokine profile and presence of EOC cells. NK cell function was evaluated by the expression of the degranulation marker CD107a in resting and interleukin (IL)-2 stimulated NK cells from ascites and blood. Degranulation of NK cells from EOC cell-free ascites was significantly (p < 0.05) higher than all the other groups, either in their resting state or after IL-2 stimulation, suggesting a previous local stimulation. In contrast, treatment with IL-2 had no effect on NK cells from ascites with EOC cells. The amount of regulatory T cells was significantly higher in ascites with EOC cells compared to EOC cell-free ascites. Ascites with EOC cells also had higher levels of tumor necrosis factor (TNF)-α, suggesting inflammation related to the malignancy. In conclusion, the functional performance of NK cells was distinct between EOC cell-free ascites and ascites with EOC cells. The impairment of NK cell response to IL-2 in ascites with EOC cells was consistent with an immunosuppressive tumor microenvironment.

Adaptive immune responses mediated by natural killer cells.

Adaptive immunity has traditionally been considered a unique feature of vertebrate physiology. Unlike innate immune responses, which remain essentially unchanged upon exposure to a recurrent challenge with the same stimulus, adaptive immune cells possess the ability to learn and remember. Thus, secondary adaptive responses to a previously encountered challenge are qualitatively and/or quantitatively distinct from those elicited by a primary encounter. Besides this capacity to acquire long-lived memory, the second cardinal feature of adaptive immunity is antigen specificity. It has been generally believed that only T and B cells can develop antigen-specific immunologic memory, because these lymphocytes uniquely express recombination-activating gene (RAG) proteins, which are necessary for somatic rearrangement of V(D)J gene segments to assemble diverse antigen-specific receptors. However, recent work has uncovered discrete subsets of murine natural killer (NK) cells capable of mediating long-lived, antigen-specific recall responses to a variety of hapten-based contact sensitizers. These NK cells appear to use distinct, RAG-independent mechanisms to generate antigen specificity. Murine NK cells have also recently been shown to develop memory upon viral infection. Here, we review recent evidence indicating that at least some NK cells are capable of mediating what appears to be adaptive immunity and discuss potential mechanisms that may contribute to RAG-independent generation of antigenic diversity and longevity.

Natural killer cell-mediated contact sensitivity develops rapidly and depends on interferon-α, interferon-γ and interleukin-12.

Natural killer (NK) cell-mediated contact sensitivity was recently described in mice. Here, we confirm NK cell-mediated contact sensitivity (CS) in SCID and RAG1(-/-) mice but not in SCIDbeige mice, which have non-functional NK cells that lack NK cell granules. NK cell-mediated CS was transferred by liver mononuclear cells and the DX5(+) fraction of liver cells, confirming that NK cells mediate CS in the absence of T and B cells. Participation of NKT cells and B-1 cells was ruled out using Jα18(-/-) and JH(-/-) mice, respectively. Remarkably, NK cell-mediated CS was observed just 1 hr after immunization and was detectable as early as 30 min after challenge. Further, we examined cytokine requirements for NK cell-mediated CS, and found that liver mononuclear cells from interleukin-12(-/-) , interferon-γ(-/-) and interferon-α receptor(-/-) donors fail to transfer NK cell-mediated CS to naive hosts. Our studies clearly show that dinitrofluorobenzene sensitized NK cells mediate very rapid, antigen-specific cell-mediated immunity, with features of both innate and acquired immune responses.

Natural killer cells for antiviral therapy.

Natural killer (NK) cell-based immunotherapy is being explored for treating infectious diseases, including viral infections. Here, we discuss evidence of NK cell responses to different viruses, ongoing clinical efforts to treat such infections with NK cell products, and review platforms to generate NK cell products.

Human Hematopoietic Stem Cell Engrafted IL-15 Transgenic NSG Mice Support Robust NK Cell Responses and Sustained HIV-1 Infection.

Mice reconstituted with human immune systems are instrumental in the investigation of HIV-1 pathogenesis and therapeutics. Natural killer (NK) cells have long been recognized as a key mediator of innate anti-HIV responses. However, established humanized mouse models do not support robust human NK cell development from engrafted human hematopoietic stem cells (HSCs). A major obstacle to human NK cell reconstitution is the lack of human interleukin-15 (IL-15) signaling, as murine IL-15 is a poor stimulator of the human IL-15 receptor. Here, we demonstrate that immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice expressing a transgene encoding human IL-15 (NSG-Tg(IL-15)) have physiological levels of human IL-15 and support long-term engraftment of human NK cells when transplanted with human umbilical-cord-blood-derived HSCs. These Hu-NSG-Tg(IL-15) mice demonstrate robust and long-term reconstitution with human immune cells, but do not develop graft-versus-host disease (GVHD), allowing for long-term studies of human NK cells. Finally, we show that these HSC engrafted mice can sustain HIV-1 infection, resulting in human NK cell responses in HIV-infected mice. We conclude that Hu-NSG-Tg(IL-15) mice are a robust novel model to study NK cell responses to HIV-1.

Sialokinin in mosquito saliva shifts human immune responses towards intracellular pathogens.

Mosquito saliva is a mix of numerous proteins that are injected into the skin while the mosquito searches for a blood meal. While mosquito saliva is known to be immunogenic, the salivary components driving these immune responses, as well as the types of immune responses that occur, are not well characterized. We investigated the effects of one potential immunomodulatory mosquito saliva protein, sialokinin, on the human immune response. We used flow cytometry to compare human immune cell populations between humanized mice bitten by sialokinin knockout mosquitoes or injected with sialokinin, and compared them to those bitten by wild-type mosquitoes, unbitten, or saline-injected control mice. Humanized mice received 4 mosquito bites or a single injection, were euthanized after 7 days, and skin, spleen, bone marrow, and blood were harvested for immune cell profiling. Our results show that bites from sialokinin knockout mosquitoes induced monocyte and macrophage populations in the skin, blood, bone marrow, and spleens, and primarily affected CD11c- cell populations. Other increased immune cells included plasmacytoid dendritic cells in the blood, natural killer cells in the skin and blood, and CD4+ T cells in all samples analyzed. Conversely, we observed that mice bitten with sialokinin knockout mosquitoes had decreased NKT cell populations in the skin, and fewer B cells in the blood, spleen, and bone marrow. Taken together, we demonstrated that sialokinin knockout saliva induces elements of a TH1 cellular immune response, suggesting that the sialokinin peptide is inducing a TH2 cellular immune response during wild-type mosquito biting. These findings are an important step towards understanding how mosquito saliva modulates the human immune system and which components of saliva may be critical for arboviral infection. By identifying immunomodulatory salivary proteins, such as sialokinin, we can develop vaccines against mosquito saliva components and direct efforts towards blocking arboviral infections.

Distinct baseline immune characteristics associated with responses to conjugated and unconjugated pneumococcal polysaccharide vaccines in older adults.

Pneumococcal infections cause serious illness and death among older adults. A capsular polysaccharide vaccine PPSV23 (Pneumovax®) and a conjugated polysaccharide vaccine PCV13 (Prevnar®) are used to prevent these infections, yet underlying responses, and baseline predictors remain unknown. We recruited and vaccinated 39 older adults (>60 years) with PPSV23 or PCV13. Both vaccines induced strong antibody responses at day 28 and similar plasmablast transcriptional signatures at day 10, however, their baseline predictors were distinct. Analyses of baseline flow cytometry and RNA-seq data (bulk and single cell) revealed a novel baseline phenotype that is specifically associated with weaker PCV13 responses, characterized by i) increased expression of cytotoxicity-associated genes and increased CD16+ NK frequency; ii) increased Th17 and decreased Th1 cell frequency. Men were more likely to display this cytotoxic phenotype and mounted weaker responses to PCV13 than women. Baseline expression levels of a distinct gene set was predictive of PPSV23 responses. This first precision vaccinology study for pneumococcal vaccine responses of older adults uncovered novel and distinct baseline predictors that might transform vaccination strategies and initiate novel interventions.

CXCR6+ and NKG2C+ Natural Killer Cells Are Distinct With Unique Phenotypic and Functional Attributes Following Bone Marrow Transplantation.

Reactivation of human cytomegalovirus (HCMV) is a life-threatening complication in transplant patients. Natural Killer (NK) cells are the first lymphocyte lineage to reconstitute following an allogeneic hematopoietic stem cell transplant (HSCT). Amongst them, NK cell Group 2 isoform C/Killer cell lectin-like receptor subfamily C, member 2 (NKG2C)-expressing NK cells contribute significantly to patient protection upon HCMV reactivation. NKG2C+ NK cells are capable of immunological memory, albeit NK cell memory is not restricted to them. Hepatic C-X-C Motif Chemokine Receptor 6 (CXCR6)-expressing NK cells also mediate memory responses in mice and humans. Small numbers of them circulate and can thus be studied in peripheral blood samples. We hypothesize that NKG2C+ and CXCR6+ NK cell subsets are distinct. To test our hypothesis, we used multi-parametric flow cytometry to determine the phenotypes and effector functions of CD56bright vs. CD56dim and NKG2C+ vs. CXCR6+ human NK cell subsets in the peripheral blood (PB) of pediatric transplant recipients monthly while monitoring patients for HCMV reactivation. Interestingly, we did not find any NKG2C+CXCR6+ NK cells in the transplant recipients' peripheral blood, suggesting that NKG2C+ and CXCR6+ NK cells are distinct. Also, NKG2C-CXCR6- NK cells, rather than NKG2C+ NK cells, made up most NK cells post-transplant, even in transplant recipients with HCMV viremia. In contrast to NKG2C+ NK cells, CXCR6+ NK cells appeared phenotypically less differentiated but were highly proliferative and produced IFN-γ and TNF α . Our findings contribute to our understanding of post-transplant NK cell development and its implications for human health.

Hematopoietic stem and progenitor cells improve survival from sepsis by boosting immunomodulatory cells.

New therapeutic strategies to reduce sepsis-related mortality are urgently needed, as sepsis accounts for one in five deaths worldwide. Since hematopoietic stem and progenitor cells (HSPCs) are responsible for producing blood and immune cells, including in response to immunological stress, we explored their potential for treating sepsis. In a mouse model of Group A Streptococcus (GAS)-induced sepsis, severe immunological stress was associated with significant depletion of bone marrow HSPCs and mortality within approximately 5-7 days. We hypothesized that the inflammatory environment of GAS infection drives rapid HSPC differentiation and depletion that can be rescued by infusion of donor HSPCs. Indeed, infusion of 10,000 naïve HSPCs into GAS-infected mice resulted in rapid myelopoiesis and a 50-60% increase in overall survival. Surprisingly, mice receiving donor HSPCs displayed a similar pathogen load compared to untreated mice. Flow cytometric analysis revealed a significantly increased number of myeloid-derived suppressor cells in HSPC-infused mice, which correlated with reduced inflammatory cytokine levels and restored HSPC levels. These findings suggest that HSPCs play an essential immunomodulatory role that may translate into new therapeutic strategies for sepsis.