HIV-1 Nef-mediated downregulation of CD155 results in viral restriction by KIR2DL5+ NK cells.

Antiviral NK cell activity is regulated through the interaction of activating and inhibitory NK cell receptors with their ligands on infected cells. HLA class I molecules serve as ligands for most killer cell immunoglobulin-like receptors (KIRs), but no HLA class I ligands for the inhibitory NK cell receptor KIR2DL5 have been identified to date. Using a NK cell receptor/ligand screening approach, we observed no strong binding of KIR2DL5 to HLA class I or class II molecules, but confirmed that KIR2DL5 binds to the poliovirus receptor (PVR, CD155). Functional studies using primary human NK cells revealed a significantly decreased degranulation of KIR2DL5+ NK cells in response to CD155-expressing target cells. We subsequently investigated the role of KIR2DL5/CD155 interactions in HIV-1 infection, and showed that multiple HIV-1 strains significantly decreased CD155 expression levels on HIV-1-infected primary human CD4+ T cells via a Nef-dependent mechanism. Co-culture of NK cells with HIV-1-infected CD4+ T cells revealed enhanced anti-viral activity of KIR2DL5+ NK cells against wild-type versus Nef-deficient viruses, indicating that HIV-1-mediated downregulation of CD155 renders infected cells more susceptible to recognition by KIR2DL5+ NK cells. These data show that CD155 suppresses the antiviral activity of KIR2DL5+ NK cells and is downmodulated by HIV-1 Nef protein as potential trade-off counteracting activating NK cell ligands, demonstrating the ability of NK cells to counteract immune escape mechanisms employed by HIV-1

HIV-1 induced changes in HLA-C*03 : 04-presented peptide repertoires lead to reduced engagement of inhibitory natural killer cell receptors

OBJECTIVE: Viral infections influence intracellular peptide repertoires available for presentation by HLA-I. Alterations in HLA-I/peptide complexes can modulate binding of killer immunoglobuline-like receptors (KIRs) and thereby the function of natural killer (NK) cells. Although multiple studies have provided evidence that HLA-I/KIR interactions play a role in HIV-1 disease progression, the consequence of HIV-1 infection for HLA-I/KIR interactions remain largely unknown. DESIGN: We determined changes in HLA-I presented peptides resulting from HIV-1-infection of primary human CD4 T cells and assessed the impact of changes in peptide repertoires on HLA-I/KIR interactions. METHODS: Liquid chromatography-coupled tandem mass spectrometry to identify HLA-I presented peptides, cell-based in-vitro assays to evaluate functional consequences of alterations in immunopeptidome and atomistic molecular dynamics simulations to confirm experimental data. RESULTS: A total of 583 peptides exclusively presented on HIV-1-infected cells were identified, of which only 0.2% represented HIV-1 derived peptides. Focusing on HLA-C*03 : 04/KIR2DL3 interactions, we observed that HLA-C*03 : 04-presented peptides derived from noninfected CD4 T cells mediated stronger binding of inhibitory KIR2DL3 than peptides derived from HIV-1-infected cells. Furthermore, the most abundant peptide presented by HLA-C*03 : 04 on noninfected CD4 T cells (VIYPARISL) mediated the strongest KIR2DL3-binding, while the most abundant peptide presented on HIV-1-infected cells (YAIQATETL) did not mediate KIR2DL3-binding. Molecular dynamics simulations of HLA-C*03 : 04/KIR2DL3 interactions in the context of these two peptides revealed that VIYPARISL significantly enhanced the HLA-C*03 : 04/peptide contact area to KIR2DL3 compared with YAIQATETL. CONCLUSION: These data demonstrate that HIV-1 infection-induced changes in HLA-I-presented peptides can reduce engagement of inhibitory KIRs, providing a mechanism for enhanced activation of NK cells by virus-infected cells

CD49a Expression Identifies a Subset of Intrahepatic Macrophages in Humans

Macrophages play central roles in inflammatory reactions and initiation of immune responses during infections. More than 80% of total tissue macrophages are described to be located in the liver as liver-resident macrophages, also named Kupffer cells (KCs). While studies in mice have established a central role of liver-resident KCs in regulating liver inflammation, their phenotype and function are not well-characterized in humans. Comparing paired human liver and peripheral blood samples, we observed significant differences in the distribution of macrophage (Mφ) subsets, with lower frequencies of CD14hiCD16lo and higher frequencies of CD14int−hiCD16int Mφ in human livers. Intrahepatic Mφ consisted of diverse subsets with differential expression of CD49a, a liver-residency marker previously described for human and mice NK cells, and VSIG4 and/or MARCO, two recently described human tissue Mφ markers. Furthermore, intrahepatic CD49a+ Mφ expressed significantly higher levels of maturation and activation markers, exhibited higher baseline levels of TNF-α, IL-12, and IL-10 production, but responded less to additional in vitro TLR stimulation. In contrast, intrahepatic CD49a− Mφ were highly responsive to stimulation with TLR ligands, similar to what was observed for CD49a− monocytes (MOs) in peripheral blood. Taken together, these studies identified populations of CD49a+, VSIG4+, and/or MARCO+ Mφ in human livers, and demonstrated that intrahepatic CD49a+ Mφ differed in phenotype and function from intrahepatic CD49a− Mφ as well as from peripheral blood-derived monocytes

Unsupervised analysis of intrahepatic CD49a+ and CD49a- NK cells.

<p><b>(A)</b> Gated CD49a+ and <b>(B)</b> CD49a- NK cells from 19 donors were concatenated and represented in t-SNE maps for the expression of chemokine receptors, activation and residency markers. Color coding indicates the expression intensity of the surface marker, pink being higher expressed and green being lower expressed.</p

Proliferative capacity exhibited by human liver-resident CD49a+CD25+ NK cells

<div><p>The recruitment and retention of Natural Killer (NK) cells in the liver are thought to play an important role during hepatotropic infections and liver cirrhosis. The aims of this study were to determine differences between liver-derived and peripheral blood-derived NK cells in the context of liver inflammation and cirrhosis. We conducted a prospective dual-center cross-sectional study in patients undergoing liver transplantation or tumor-free liver resections, in which both liver tissue and peripheral blood samples were obtained from each consenting study participants. Intrahepatic lymphocytes and PBMCs were stained, fixed and analyzed by flow cytometry. Our results showed that, within cirrhotic liver samples, intrahepatic NK cells were particularly enriched for CD49a+ NK cells when compared to tumor-free liver resection samples. CD49a+ liver-derived NK cells included populations of cells expressing CD25, CD34 and CXCR3. Moreover, CD49a+CD25+ liver-derived NK cells exhibited high proliferative capacity <i>in vitro</i> in response to low doses of IL-2. Our study identified a specific subset of CD49a+CD25+ NK cells in cirrhotic livers bearing functional features of proliferation.</p></div

Unsupervised analysis of intrahepatic CD49a+ and CD49a- NK cells.

<p><b>(A)</b> Gated CD49a+ and <b>(B)</b> CD49a- NK cells from 19 donors were concatenated and represented in t-SNE maps for the expression of chemokine receptors, activation and residency markers. Color coding indicates the expression intensity of the surface marker, pink being higher expressed and green being lower expressed.</p

Demographics and clinical characteristics from explanted liver tissue samples.

<p>Demographics and clinical characteristics from explanted liver tissue samples.</p

Immune phenotyping of combined peripheral and intrahepatic NK cells.

<p>Gated NK cells from 19 donors were concatenated and represented in t-SNE maps for the expression of chemokine receptors, activation and residency markers. <b>(A)</b> peripheral and <b>(B)</b> intrahepatic NK cells are shown. Color coding indicates the expression intensity of the surface marker, pink being higher expressed and green being lower expressed. <b>(C)</b> Proportion of NK cells derived from the liver (ihNK) and the peripheral blood (pNK) on the liver transplantation cohort expressing CD49a (pNK median (IQR): 0.9 (0.3–3.9); ihNK median (IQR): 34.4 (27.6–40.5); p<0.0001), CD34 (pNK median (IQR): 2.2 (1–4.7); ihNK median (IQR): 12 (6.8–20.9); p<0.0001), CXCR4 (pNK median (IQR): 9.8 (4.9–22.2); ihNK median (IQR): 3.4 (1.3–7.7); p = 0.0024), CD57 (pNK median (IQR): 19 (22–38.5); ihNK median (IQR): 13.7 (9.4–23.3); p<0.0001) and DNAM-1 (pNK median (IQR): 79.6 (51.5–85.6); ihNK median (IQR): 26.5 (8.5–32.1); p<0.0001) (n = 19). <b>(D)</b> Proportion of NK cells from the tumor-free liver resections expressing CD49a, CD34, CD57, DNAM-1, CXCR3 and CXCR4 within the IHLs NK cells and pNK cells (n = 5). <b>(E)</b> Frequency of CD49a+ NK cell population within the IHLs NK cells in tumor-free liver resection cohort (HLR) and the liver retransplant cohort (cirrhotic livers, CL). Data is depicted as scatter plot, with each dot corresponding to a participant. Bars indicate median and IQR. Wilcoxon signed rank tests with adjustment of p-values by false discovery rate.</p

Relevance of CD49a+CD25+ ihNK cells.

<p><b>(A)</b> Boolean gating of CD49a, CD25 and CD34 markers on ihNK cells. <b>(B)</b> Pie chart representing the frequency of each of the 7 possible combinations of the 3 markers, CD49a, CD25 and CD34. The median percentage of each population is represented. <b>(C)</b> Alanine Aminotransferase (ALT) serum levels correlation with the proportion of intrahepatic CD49a+CD25+ NK cells in the liver transplantation cohort. Data in <b>(A)</b> is depicted as scatter plot showing all individuals, the bar represents the median and the deviation is depicted as interquartile range.</p