The CD56-CD16+ NK cell subset in chronic infections.

Long-term human diseases can shape the immune system, and natural killer (NK) cells have been documented to differentiate into distinct subsets specifically associated with chronic virus infections. One of these subsets found in large frequencies in HIV-1 are the CD56-CD16+ NK cells, and this population's association with chronic virus infections is the subject of this review. Human NK cells are classically defined by CD56 expression, yet increasing evidence supports the NK cell status of the CD56-CD16+ subset which we discuss herein. We then discuss the evidence linking CD56-CD16+ NK cells to chronic virus infections, and the potential immunological pathways that are altered by long-term infection that could be inducing the population's differentiation. An important aspect of NK cell regulation is their interaction with human leukocyte antigen (HLA) class-I molecules, and we highlight work that indicates both virus and genetic-mediated variations in HLA expression that have been linked to CD56-CD16+ NK cell frequencies. Finally, we offer a perspective on CD56-CD16+ NK cell function, taking into account recent work that implies the subset is comparable to CD56+CD16+ NK cell functionality in antibody-dependent cell cytotoxicity response, and the definition of CD56-CD16+ NK cell subpopulations with varying degranulation capacity against target cells.

Roles of N-linked glycosylation and glycan-binding proteins in placentation: trophoblast infiltration, immunomodulation, angiogenesis, and pathophysiology.

Protein N-linked glycosylation is a structurally diverse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a diverse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins. Evidence exists to suggest that altering the structural nature of their N-glycans can impact several trophoblast functions, which include those related to interactions with decidual cells. This review summarizes trophoblast-related activities influenced by N-glycan-GBP recognition, exploring how different subtypes of trophoblasts actively adapt to characteristics of the decidualized endometrium through cell-specific expression of N-glycosylated proteins, and how these cells receive decidua-derived signals via N-glycan-GBP interactions. We highlight work on how changes in N-glycosylation relates to the success of trophoblast infiltration, interactions of immunomodulators, and uterine angiogenesis. We also discuss studies that suggest aberrant N-glycosylation of trophoblasts may contribute to the pathogenesis of pregnancy complications (e.g. pre-eclampsia, early spontaneous miscarriages and hydatidiform mole). We propose that a more in-depth understanding of how N-glycosylation shapes trophoblast phenotype during early pregnancy has the potential to improve our approach to predicting, diagnosing and alleviating poor maternal/fetal outcomes associated with placental dysfunction.

The role of CD8+ T-cell clones in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multidimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterized patients with ITP and compared them with age-matched controls using immunophenotyping, next-generation sequencing of T-cell receptor (TCR) genes, single-cell RNA sequencing, and functional T-cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon gamma, tumor necrosis factor α, and granzyme B, defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the TCR showed expanded T-cell clones in patients with ITP. T-cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon gamma, and trigger platelet activation and apoptosis via the TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP.

CD56-negative NK cells: Frequency in peripheral blood, expansion during HIV-1 infection, functional capacity, and KIR expression.

Human NK cells are usually defined as CD3-CD56+ lymphocytes. However, a CD56-CD16+ (CD56neg) lymphocyte population that displays NK-associated markers expands during chronic viral infections such as HIV-1 and HCV, and, to lesser extent, in herpesvirus infections. This CD56neg NK cell subset has been understudied because it requires the exclusion of other lymphocytes to accurately identify its presence. Many questions remain regarding the origin, development, phenotype, and function of the CD56neg NK cell population. Our objective was to determine the frequency of this NK subset in healthy controls and its alteration in viral infections by performing a meta-analysis. In addition to this, we analyzed deposited CyTOF and scRNAseq datasets to define the phenotype and subsets of the CD56neg NK cell population, as well as their functional variation. We found in 757 individuals, from a combined 28 studies and 6 datasets, that the CD56neg subset constitutes 5.67% of NK cells in healthy peripheral blood, while HIV-1 infection increases this population by a mean difference of 10.69%. Meta-analysis of surface marker expression between NK subsets showed no evidence of increased exhaustion or decreased proliferation within the CD56neg subset. CD56neg NK cells have a distinctive pattern of KIR expression, implying they have a unique potential for KIR-mediated education. A perforin-CD94-NKG2C-NKp30- CD56neg population exhibited different gene expression and degranulation responses against K562 cells compared to other CD56neg cells. This analysis distinguishes two functionally distinct subsets of CD56neg NK cells. They are phenotypically diverse and have differing capacity for education by HLA class-I interactions with KIRs.

Natural LILRB1 D1-D2 variants show frequency differences in populations and bind to HLA class I with various avidities.

Leukocyte immunoglobulin-like receptor B1 (LILRB1) is widely expressed on various immune cells and the engagement of LILRB1 to HLA class I and pathogen-derived proteins can modulate the immune response. In the current study, 108 LILRB1 alleles were identified by screening the LILRB1 locus from the 1000 Genomes Phase 3 database. Forty-six alleles that occurred in three or more individuals encode 28 LILRB1 allotypes, and the inferred LILRB1 allotypes were then grouped into 9 LILRB1 D1-D2 variants for further analysis. We found that variants 1, 2, and 3 represent the three most frequent LILRB1 D1-D2 variants and the nine variants show frequency differences in populations. The binding assay demonstrated that variant 1 bound to HLA class I with the highest avidity, and all tested LILRB1 D1-D2 variants bound to HLA-C with lower avidity than to HLA-A and -B. Locus-specific polymorphisms at positions 183, 189, and 268 in HLA class I and dimorphisms in HLA-A (positions 207 and 253) and in HLA-B (position 194) affect their binding to LILRB1. Notably, the electrostatic interaction plays a critical role in the binding of LILRB1 to HLA class I as revealed by electrostatic analysis and by comparison of different binding avidities caused by polymorphisms at positions 72 and 103 of LILRB1. In this paper, we present a comprehensive study of the population genetics and binding abilities of LILRB1. The data will help us better understand the LILRB1-related diversity of the immune system and lay a foundation for functional studies.

Phosphoantigen-Stimulated γδ T Cells Suppress Natural Killer-Cell Responses to Missing-Self.

γδ T cells stimulated by phosphoantigens (pAg) are potent effectors that secrete Th1 cytokines and kill tumor cells. Consequently, they are considered candidates for use in cancer immunotherapy. However, they have proven only moderately effective in several clinical trials. We studied the consequences of pAg-stimulated γδ T-cell interactions with natural killer (NK) cells and CD8+ T cells, major innate and adaptive effectors, respectively. We found that pAg-stimulated γδ T cells suppressed NK-cell responses to "missing-self" but had no effect on antigen-specific CD8+ T-cell responses. Extensive analysis of the secreted cytokines showed that pAg-stimulated γδ T cells had a proinflammatory profile. CMV-pp65-specific CD8+ T cells primed with pAg-stimulated γδ T cells showed little effect on responses to pp65-loaded target cells. By contrast, NK cells primed similarly with γδ T cells had impaired capacity to degranulate and produce IFNγ in response to HLA class I-deficient targets. This effect depended on BTN3A1 and required direct contact between NK cells and γδ T cells. γδ T-cell priming of NK cells also led to a downregulation of NKG2D and NKp44 on NK cells. Every NK-cell subset was affected by γδ T cell-mediated immunosuppression, but the strongest effect was on KIR+NKG2A- NK cells. We therefore report a previously unknown function for γδ T cells, as brakes of NK-cell responses to "missing-self." This provides a new perspective for optimizing the use of γδ T cells in cancer immunotherapy and for assessing their role in immune responses to pAg-producing pathogens. See related Spotlight by Kabelitz, p. 543.

Isolation of single cells from human uterus in the third trimester of pregnancy: myometrium, decidua, amnion and chorion.

During pregnancy, interactions between uterine immune cells and cells of the surrounding reproductive tissues are thought to be vital for regulating labour. The mechanism that specifically initiates spontaneous labour has not been determined, but distinct changes in uterine immune cell populations and their activation status have been observed during labour at term gestation. To understand the regulation of human labour by the immune system, the ability to isolate both immune cells and non-immune cells from the uterus is required. Here, we describe protocols developed in our laboratory to isolate single cells from uterine tissues, which preserve both immune and non-immune cell populations for further analysis. We provide detailed methods for isolating immune and non-immune cells from human myometrium, chorion, amnion and decidua, together with representative flow cytometry analysis of isolated cell populations present. The protocols can be completed in tandem and take approximately 4-5 h, resulting in single-cell suspensions that contain viable leucocytes, and non-immune cells in sufficient numbers for single-cell analysis approaches such as flow cytometry and single cell RNA sequencing (scRNAseq).

Pregnancy Gestation Impacts on HIV-1-Specific Granzyme B Response and Central Memory CD4 T Cells.

Pregnancy induces alterations in peripheral T-cell populations with both changes in subset frequencies and anti-viral responses found to alter with gestation. In HIV-1 positive women anti-HIV-1 responses are associated with transmission risk, however detailed investigation into both HIV-1-specific memory responses associated with HIV-1 control and T-cell subset changes during pregnancy have not been undertaken. In this study we aimed to define pregnancy and gestation related changes to HIV-1-specific responses and T-cell phenotype in ART treated HIV-1 positive pregnant women. Eleven non-pregnant and 24 pregnant HIV-1 positive women were recruited, peripheral blood samples taken, fresh cells isolated, and compared using ELISpot assays and flow cytometry analysis. Clinical data were collected as part of standard care, and non-parametric statistics used. Alterations in induced IFNγ, IL-2, IL-10, and granzyme B secretion by peripheral blood mononuclear cells in response to HIV-1 Gag and Nef peptide pools and changes in T-cell subsets between pregnant and non-pregnant women were assessed, with data correlated with participant clinical parameters and longitudinal analysis performed. Cross-sectional comparison identified decreased IL-10 Nef response in HIV-1 positive pregnant women compared to non-pregnant, while correlations exhibited reversed Gag and Nef cytokine and protease response associations between groups. Longitudinal analysis of pregnant participants demonstrated transient increases in Gag granzyme B response and in the central memory CD4 T-cell subset frequency during their second trimester, with a decrease in CD4 effector memory T cells from their second to third trimester. Gag and Nef HIV-1-specific responses diverge with pregnancy time-point, coinciding with relevant T-cell phenotype, and gestation associated immunological adaptations. Decreased IL-10 Nef and both increased granzyme B Gag response and central memory CD4 T cells implies that amplified antigen production is occurring, which suggests a period of compromised HIV-1 control in pregnancy.

Short Communication: Therapeutic Immunization Benefits Mucosal-Associated Invariant T Cell Recovery in Contrast to Interleukin-2, Granulocyte-Macrophage Colony-Stimulating Factor, and Recombinant Human Growth Hormone Addition in HIV-1+ Treated Patients: Individual Case Reports from Phase I Trial.

Mucosal-associated invariant T (MAIT) cell populations are reduced in frequency in HIV-1+ patients, and this disruption is associated with systemic immune activation. Reconstitution of MAIT frequency may benefit HIV-1-infected individuals; however, only recently has in vivo work been endeavored. Treatment with interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), and recombinant human growth hormone (rhGH) immunotherapy combined with an HIV-1 vaccine in the context of antiretroviral therapy (ART) has shown to reconstitute CD4 T cell population numbers and function. In this study cryopreserved peripheral blood mononuclear cells (PBMCs) from 12 HIV-1+ patients who were undergoing a combination of HIV-1 vaccine and/or IL-2, GM-CSF and rhGH immunotherapy in conjunction with ART were analyzed to assess the potential of this treatment to promote MAIT cell proliferation. PBMCs were thawed from study baseline, weeks 2 and 48 time points, fluorescently stained for MAIT cell markers, and assessed by flow cytometric analysis. Matched pairs and intergroup results were statistically compared using appropriate methods. MAIT cell frequency was increased from baseline at 48 weeks in participants who received vaccine only, whereas individuals receiving IL-2, GM-CSF, and rhGH immunotherapy with or without vaccine did not show additional benefit. Although IL-2, GM-CSF, and rhGH treatment promotes CD4 T cell reconstitution and HIV-1-specific T cell function, it does not support MAIT cell recovery in patients on suppressive ART. Therapeutic immunization however has a positive effect, highlighting the importance of aiming for balanced promotion of T cell population reconstitution to impact on HIV-1 transmission and pathogenesis.

Enrichment of HLA Types and Single-Nucleotide Polymorphism Associated With Non-progression in a Strictly Defined Cohort of HIV-1 Controllers.

HIV-1 controllers (HIC) are extremely rare patients with the ability to control viral replication, maintain unchanging CD4 T-cell count, and evade disease progression for extensive periods of time, in the absence of antiretroviral therapy. In order to establish the representation of key genetic correlates of atypical disease progression within a cohort of HIV-1+ individuals who control viral replication, we examine four-digit resolution HLA type and single-nucleotide polymorphisms (SNP) previously identified to be correlated to non-progressive infection, in strictly defined HIC. Clinical histories were examined to identify patients exhibiting HIC status. Genomic DNA was extracted, and high definition HLA typing and genome-wide SNP analysis was performed. Data were compared with frequencies of SNP in European long-term non-progressors (LTNP) and primary infection cohorts. HLA-B alleles associated with atypical disease progression were at very high frequencies in the group of five HIC studied. All four HIC of European ancestry were HLA-B*57+ and half were also HLA-B*27+. All HIC, including one of self-reported African ethnicity, had the HLA-Cw*0602 allele, and the HLA-DQ9 allele was present only in HIC of European ancestry. A median 95% of the top 19 SNP known to be associated with LTNP status was observed in European HIC (range 78-100%); 17/19 of the SNP considered mapped to chromosome 6 in the HLA region, whereas 2/19 mapped to chromosome 8. The HIC investigated here demonstrated high enrichment of HLA types and SNP previously associated with long-term non-progression. These findings suggest that the extreme non-progressive phenotype considered here is associated with a genetic signature characterized by a single-genetic unit centered around the HLA-B*57 haplotype and the possible additive effect of HLA-B*27.