Henoch S. Hong

Cytokine-activated NK cells inhibit PMN apoptosis and preserve their functional capacity

Natural killer (NK) cells and polymorphonuclear cells (PMNs) play a critical role in the first line of defense against microorganisms. Upon host infection, PMNs phagocytose invading pathogens with subsequent killing by oxidative or nonoxidative mechanisms. NK cells are known to have immunoregulatory effects on T cells, B cells, dendritic cells (DCs), and monocytes through secretion of various soluble products and cell-cell contact. However, their impact on PMN survival and function is not well known. We found that soluble factors derived from cytokine-activated NK cells delay PMN apoptosis and preserve their ability to perform phagocytosis and produce reactive oxygen species (ROS). The expression patterns of CD11b and CD62L on PMNs differed according to the cytokine combination used for NK-cell stimulation. Irrespective of the NK-cell treatment, however, PMN survival was prolonged with sustained functional capacity. We found that interferon gamma, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha produced by NK cells upon stimulation with cytokines played a crucial role in NK cell-mediated effects on PMNs. Our study demonstrates that soluble factors derived from cytokine-activated NK cells send survival signals to PMNs, which would promote their accumulation and function at the site of inflammation in vivo.

Phenotypically and functionally distinct subsets contribute to the expansion of CD56-/CD16+ natural killer cells in HIV infection.

Objective:Chronic HIV infection has been associated with activation and increased turnover of natural killer (NK) cells as well as with disturbed homeostasis of the NK cell compartment, including loss of CD56+ NK cells and accumulation of dysfunctional CD56−/CD16+ NK cells. We performed a comprehensive phenotypical and functional characterization of this population. Design:A cross-sectional study was performed to analyze CD56−/CD16+ NK cells from 34 untreated HIV-infected and 15 seronegative individuals. Methods:NK cells were analyzed by flow cytometry. Degranulation was assessed by measuring their expression of CD107a after stimulation with K562 cells, interleukin-12 and interleukin-15. Results:CD56−/CD16+ NK cells are heterogeneous and composed of two populations, namely CD122−/CCR7+ cells and CD122+/CCR7− cells. We show that expanded CD122+ but not CCR7+ cells in HIV-seropositive individuals are characterized by expression of senescence marker CD57 similarly to CD56dim/CD16+ NK cells along with expression of KIRs, CD8, perforin and granzyme B. Despite expression of perforin and granzyme B, CD57 expressing cells exhibited less numbers of degranulating cells as measured by CD107a, indicating their functional impairment. However, there was no correlation between expansion of total CD56−/CD16+ NK cells or the distinct subpopulations and viral load or CD4 cell count. Conclusion:These data indicate that expansion of CD56−/CD16+ cells in HIV infection is driven by a distinct subset within this population with high expression of terminal differentiation marker with a phenotype resembling CD56dim/CD16+ NK cells.

Loss of CCR7 Expression on CD56 bright NK Cells Is Associated with a CD56 dim CD16 + NK Cell-Like Phenotype and Correlates with HIV Viral Load

NK cells are pivotal sentinels of the innate immune system and distinct subpopulations in peripheral blood have been described. A number of studies addressed HIV-induced alterations of NK cell phenotype and functionality mainly focusing on CD56dimCD16+ and CD56−CD16+ NK cells. However, the impact of HIV-infection on CD56bright NK cells is less well understood. Here we report a rise of CD56bright NK cells in HIV-infected individuals, which lack CCR7-expression and strongly correlate with HIV viral load. CCR7−CD56bright NK cells were characterized by increased cytolytic potential, higher activation states and a more differentiated phenotype. These cells thus acquired a number of features of CD56dimCD16+ NK cells. Furthermore, CD56bright NK cells from HIV patients exhibited higher degranulation levels compared to uninfected individuals. Thus, chronic HIV-infection is associated with a phenotypic and functional shift of CD56bright NK cells, which provides a novel aspect of HIV-associated pathogenesis within the NK cell compartment.

Safety and immunogenicity of a mRNA rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first-in-human phase 1 clinical trial

BACKGROUND Vaccines based on mRNA coding for antigens have been shown to be safe and immunogenic in preclinical models. We aimed to report results of the first-in-human proof-of-concept clinical trial in healthy adults of a prophylactic mRNA-based vaccine encoding rabies virus glycoprotein (CV7201). METHODS We did an open-label, uncontrolled, prospective, phase 1 clinical trial at one centre in Munich, Germany. Healthy male and female volunteers (aged 18-40 years) with no history of rabies vaccination were sequentially enrolled. They received three doses of CV7201 intradermally or intramuscularly by needle-syringe or one of three needle-free devices. Escalating doses were given to subsequent cohorts, and one cohort received a booster dose after 1 year. The primary endpoint was safety and tolerability. The secondary endpoint was to determine the lowest dose of CV7201 to elicit rabies virus neutralising titres equal to or greater than the WHO-specified protective antibody titre of 0·5 IU/mL. The study is continuing for long-term safety and immunogenicity follow-up. This trial is registered with ClinicalTrials.gov, number NCT02241135. FINDINGS Between Oct 21, 2013, and Jan 11, 2016, we enrolled and vaccinated 101 participants with 306 doses of mRNA (80-640 μg) by needle-syringe (18 intradermally and 24 intramuscularly) or needle-free devices (46 intradermally and 13 intramuscularly). In the 7 days post vaccination, 60 (94%) of 64 intradermally vaccinated participants and 36 (97%) of 37 intramuscularly vaccinated participants reported solicited injection site reactions, and 50 (78%) of 64 intradermally vaccinated participants and 29 (78%) of 37 intramuscularly vaccinated participants reported solicited systemic adverse events, including ten grade 3 events. One unexpected, possibly related, serious adverse reaction that occurred 7 days after a 640 μg intramuscular dose resolved without sequelae. mRNA vaccination by needle-free intradermal or intramuscular device injection induced virus neutralising antibody titres of 0·5 IU/mL or more across dose levels and schedules in 32 (71%) of 45 participants given 80 μg or 160 μg CV7201 doses intradermally and six (46%) of 13 participants given 200 μg or 400 μg CV7201 doses intramuscularly. 1 year later, eight (57%) of 14 participants boosted with an 80 μg needle-free intradermal dose of CV7201 achieved titres of 0·5 IU/mL or more. Conversely, intradermal or intramuscular needle-syringe injection was ineffective, with only one participant (who received 320 μg intradermally) showing a detectable immune response. INTERPRETATION This first-ever demonstration in human beings shows that a prophylactic mRNA-based candidate vaccine can induce boostable functional antibodies against a viral antigen when administered with a needle-free device, although not when injected by a needle-syringe. The vaccine was generally safe with a reasonable tolerability profile. FUNDING CureVac AG.

Characterization of CD8 + T Cell Differentiation following SIVΔnef Vaccination by Transcription Factor Expression Profiling

The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8+ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8++ T cells, and in SIV-specific CD8+ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8+ T cells into groups concordant with cell surface phenotype. Transcription factor expression patterns in SIV-specific CD8+ T cells in SIVΔnef-vaccinated animals were distinct from those observed in purified CD8+ T cell subsets obtained from naïve animals, and were intermediate to expression profiles of purified central memory and effector memory T cells. Expression of transcription factors elicited by SIVΔnef vaccination also varied over time: cells obtained at later time points, temporally associated with greater protection, appeared more central-memory like than cells obtained at earlier time points, which appeared more effector memory-like. Expression of transcription factors associated with effector differentiation, such as ID2 and RUNX3, were decreased over time, while expression of transcription factors associated with quiescence or memory differentiation, such as TCF7, BCOR and EOMES, increased. CD8+ T cells specific for a more conserved epitope expressed higher levels of TBX21 and BATF, and appeared more effector-like than cells specific for an escaped epitope, consistent with continued activation by replicating vaccine virus. These data suggest transcription factor expression profiling is a novel method that can provide additional data complementary to the analysis of memory cell differentiation based on classical phenotypic markers. Additionally, these data support the hypothesis that ongoing stimulation by SIVΔnef promotes a distinct protective balance of CD8+ T cell differentiation and activation states.

High Frequencies of Polyfunctional CD8+ NK Cells in Chronic HIV-1 Infection Are Associated with Slower Disease Progression

ABSTRACT Natural killer (NK) cells are effector and regulatory innate immune cells and play a critical role in the first line of defense against various viral infections. Although previous reports have indicated the vital contributions of NK cells to HIV-1 immune control, nongenetic NK cell parameters directly associated with slower disease progression have not been defined yet. In a longitudinal, retrospective study of 117 untreated HIV-infected subjects, we show that higher frequencies as well as the absolute numbers of CD8+ CD3− lymphocytes are linked to delayed HIV-1 disease progression. We show that the majority of these cells are well-described blood NK cells. In a subsequent cross-sectional study, we demonstrate a significant loss of CD8+ NK cells in untreated HIV-infected individuals, which correlated with HIV loads and inversely correlated with CD4+ T cell counts. CD8+ NK cells had modestly higher frequencies of CD57-expressing cells than CD8− cells, but CD8+ and CD8− NK cells showed no differences in the expression of a number of activating and inhibiting NK cell receptors. However, CD8+ NK cells exhibited a more functional profile, as detected by cytokine production and degranulation. IMPORTANCE We demonstrate that the frequency of highly functional CD8+ NK cells is inversely associated with HIV-related disease markers and linked with delayed disease progression. These results thus indicate that CD8+ NK cells represent a novel NK cell-derived, innate immune correlate with an improved clinical outcome in HIV infection.

No monkey business: why studying NK cells in non-human primates pays off

Human NK (hNK) cells play a key role in mediating host immune responses against various infectious diseases. For practical reasons, the majority of the data on hNK cells has been generated using peripheral blood lymphocytes. In contrast, our knowledge of NK cells in human tissues is limited, and not much is known about developmental pathways of hNK cell subpopulations in vivo. Although research in mice has elucidated a number of fundamental features of NK cell biology, mouse, and hNK cells significantly differ in their subpopulations, functions, and receptor repertoires. Thus, there is a need for a model that is more closely related to humans and yet allows experimental manipulations. Non-human primate models offer numerous opportunities for the study of NK cells, including the study of the role of NK cells after solid organ and stem cell transplantation, as well as in acute viral infection. Macaque NK cells can be depleted in vivo or adoptively transferred in an autologous system. All of these studies are either difficult or unethical to carry out in humans. Here we highlight recent advances in rhesus NK cell research and their parallels in humans. Using high-throughput transcriptional profiling, we demonstrate that the human CD56bright and CD56dim NK cell subsets have phenotypically and functionally analogous counterparts in rhesus macaques. Thus, the use of non-human primate models offers the potential to substantially advance hNK cell research.

FcγRIII (CD16)-mediated ADCC by NK cells is regulated by monocytes and FcγRII (CD32)

Monocytes are known to engage in reciprocal crosstalk with NK cells but their influence on NK‐cell‐associated antibody‐dependent cellular cytotoxicity (ADCC) is not well understood. We demonstrate that in humans FcγRIII (CD16)‐dependent ADCC by NK cells is considerably enhanced by monocytes, and that this effect is regulated by FcγRII (CD32) crosslinking in healthy individuals. It is known that during HIV‐1 infection, NK cells are known to express low levels of CD16 and exhibit reduced ADCC. We show that immune regulation of CD16‐mediated NK‐cell cytotoxicity by monocytes through CD32 engagement is substantially disturbed in chronic progressive HIV‐1 infection. Expression of activating isoform of CD32 represented a compensatory mechanism for reduced expression of CD16 on NK cells during HIV‐1 infection. As a result, the regulation of NK‐cell‐associated ADCC by monocytes is skewed and eventually constitutes a novel factor that contributes to HIV‐1‐associated immune deficiency, dysregulation and pathogenesis. Our data therefore provide evidence, for the first time, that in humans monocytes act as a rheostat for FcγRIII‐mediated NK‐cell functions maintaining a well‐balanced immune response.

Distinct transcriptional changes in non-small cell lung cancer patients associated with multi-antigenic RNActive® CV9201 immunotherapy

ABSTRACT We recently completed a phase I/IIa trial of RNActive® CV9201, a novel mRNA-based therapeutic vaccine targeting five tumor-associated antigens in non-small cell lung cancer (NSCLC) patients. The aim of the study presented here was to comprehensively analyze changes in peripheral blood during the vaccination period and to generate hypotheses facilitating the identification of potential biomarkers correlating with differential clinical outcomes post RNActive® immunotherapy. We performed whole-genome expression profiling in a subgroup of 22 stage IV NSCLC patients before and after initiation of treatment with CV9201. Utilizing an analytic approach based on blood transcriptional modules (BTMs), a previously described, sensitive tool for blood transcriptome data analysis, patients segregated into two major clusters based on transcriptional changes post RNActive® treatment. The first group of patients was characterized by the upregulation of an expression signature associated with myeloid cells and inflammation, whereas the other group exhibited an expression signature associated with T and NK cells. Patients with an enrichment of T and NK cell modules after treatment compared to baseline exhibited significantly longer progression-free and overall survival compared to patients with an upregulation of myeloid cell and inflammatory modules. Notably, these gene expression signatures were mutually exclusive and inversely correlated. Furthermore, our findings correlated with phenotypic data derived by flow cytometry as well as the neutrophil-to-lymphocyte ratio. Our study thus demonstrates non-overlapping, distinct transcriptional profiles correlating with survival warranting further validation for the development of biomarker candidates for mRNA-based immunotherapy.

Partial recovery of senescence and differentiation disturbances in CD8+ T cell effector‐memory cells in HIV‐1 infection after initiation of ART

Immune senescence as well as disturbed CD8+ T cell differentiation are a hallmark of chronic HIV infection. Here, we investigated to what extent immune senescence is reversible after initiation of anti‐retroviral treatment (ART). Peripheral blood mononuclear cells (PBMCs) from a cohort of HIV patients with different disease courses, including untreated viral controllers (n = 10), viral non‐controllers (n = 16) and patients on ART (n = 20), were analysed and compared to uninfected controls (n = 25) by flow cytometry on bulk and HIV‐specific major histocompatibility complex (MHC) class I tetramer+ CD8+ T cells for expression of the memory markers CCR7 and CD45RO, as well as the senescence marker CD57 and the differentiation and survival marker CD127. Furthermore, a subset of patients was analysed longitudinally before and after initiation of ART. Frequencies of CD57+CD8+ T cells decreased after initiation of ART in central memory (Tcm) but not in effector memory T cell populations (TemRO and TemRA). The frequency of CD127+CD8+ cells increased in Tcm and TemRO. We observed a reduction of CD127– T cells in Tcm, TemRO and partially in TemRA subsets after initiation of ART. Importantly, HIV‐specific CD8+ TemRO cells predominantly displayed a CD127–CD57+ phenotype in untreated HIV‐patients, whereas the CD127+CD57– phenotype was under‐represented in these patients. The frequency of the CD127+CD57–CD8+ T cell subpopulation correlated strongly with absolute CD4+ counts in HIV‐infected patients before and after initiation of ART. These findings can be interpreted as a phenotypical correlate of CD8+ memory T cell differentiation and the premature ‘ageing’ of the immune system, which was even observed in successfully virally suppressed HIV patients.