Human-Induced CD49a+ NK Cells Promote Fetal Growth.

CD49a+ natural killer (NK) cells play a critical role in promoting fetal development and maintaining immune tolerance at the maternal-fetal interface during the early stages of pregnancy. However, given their residency in human tissue, thorough studies and clinical applications are difficult to perform. It is still unclear as to how functional human CD49a+ NK cells can be induced to benefit pregnancy outcomes. In this study, we established three no-feeder cell induction systems to induce human CD49a+ NK cells from umbilical cord blood hematopoietic stem cells (HSCs), bone marrow HSCs, and peripheral blood NK cells in vitro. These induced NK cells (iNKs) from three cell induction systems display high levels of CD49a, CD9, CD39, CD151 expression, low levels of CD16 expression, and no obvious cytotoxic capability. They are phenotypically and functionally similar to decidual NK cells. Furthermore, these iNKs display a high expression of growth-promoting factors and proangiogenic factors and can promote fetal growth and improve uterine artery blood flow in a murine pregnancy model in vivo. This research demonstrates the ability of human-induced CD49a+ NK cells to promote fetal growth via three cell induction systems, which could eventually be used to treat patients experiencing adverse pregnancy outcomes.

Expansions of adaptive-like NK cells with a tissue-resident phenotype in human lung and blood.

Human adaptive-like "memory" CD56dimCD16+ natural killer (NK) cells in peripheral blood from cytomegalovirus-seropositive individuals have been extensively investigated in recent years and are currently explored as a treatment strategy for hematological cancers. However, treatment of solid tumors remains limited due to insufficient NK cell tumor infiltration, and it is unknown whether large expansions of adaptive-like NK cells that are equipped for tissue residency and tumor homing exist in peripheral tissues. Here, we show that human lung and blood contains adaptive-like CD56brightCD16- NK cells with hallmarks of tissue residency, including expression of CD49a. Expansions of adaptive-like lung tissue-resident NK (trNK) cells were found to be present independently of adaptive-like CD56dimCD16+ NK cells and to be hyperresponsive toward target cells. Together, our data demonstrate that phenotypically, functionally, and developmentally distinct subsets of adaptive-like NK cells exist in human lung and blood. Given their tissue-related character and hyperresponsiveness, human lung adaptive-like trNK cells might represent a suitable alternative for therapies targeting solid tumors.

Immunological Memory in Imiquimod-Induced Murine Model of Psoriasiform Dermatitis.

Psoriasis is a common chronic inflammatory skin condition manifested by T cell responses and characterized by preferential recurrence at previously inflamed sites upon withdrawal of treatment. The site-specific disease memory in psoriasis has been linked to CD8+CD103+ tissue-resident memory T cells (Trm) in the epidermis which were previously thought to only provide "frontline" protection against pathogens and immunosurveillance during cancer development. In this study, we correlated the presence of a subset of the Trm cells which are also CD49a+ with disease severity in human psoriatic lesions with acute and chronic disease. Using an imiquimod (IMQ)-induced murine model of psoriasiform dermatitis, we also investigated the level of CD49a+ Trm cells in acute, chronic and resolved psoriatic lesions. Investigation of clinical human samples showed that patient disease severity highly correlated with the numbers of epidermal CD49a+ Trm cells. Additionally, this subset of Trm cells was shown to persist in resolved lesions of murine psoriasiform dermatitis once clinical disease features had subsided. Importantly, these CD49a+ Trm cells showed significantly higher levels of granzyme B (GzmB) production compared to acute disease, suggesting a potential role of CD49a+ Trm cells for psoriatic re-occurrence in resolved patients. Better understanding of epidermal CD49a+ Trm cell activity is necessary for development of advanced treatment strategies for psoriasis to permit long-term, continuous disease control.

Characterization of Rhesus Macaque Liver-Resident CD49a+ NK Cells During Retrovirus Infections.

CD49a+ tissue resident NK cells have been implicated in memory-like NK cell responses, but while this population is well-characterized in mice and in humans, they are poorly described in non-human primates (NHP) which are particularly critical for modeling human viral infections. Others and we have shown that memory-like NK cells are enriched in the liver and because of the importance of NHP in modeling HIV infection, understanding the immunobiology of CD49a+ NK cells in SIV-infected rhesus macaques is critical to explore the role of this cell type in retroviral infections. In this study mononuclear cells isolated from livers, spleens, and peripheral whole blood were analyzed in acutely and chronically lentivirus-infected and experimentally-naïve Indian rhesus macaques (RM). NK cells were then identified as CD45+CD14-CD20-CD3-NKG2A/C+ cells and characterized using multiparametric flow-cytometry. Our data show that in RM, CD49a+ NK cells increase in the liver following retroviral infections [median = 5.2% (naïve) vs. median = 9.48% (SIV+) or median = 16.8% (SHIV+)]. In contrast, there is little change in CD49a+ NK frequencies in whole blood or spleens of matched animals. In agreement with human and murine data we also observed that CD49a+ NK cells were predominantly Eomeslow T-betlow, though these frequencies are elevated in infected animal cohorts. Functionally, our data suggests that infection alters TNF-α, IFN-γ, and CD107a expression in stimulated CD49a+ NK cells. Specifically, our analyses found a decrease in CD49a+ CD107a+ TNFα+ IFNγ- NK cells, with a simultaneous increase in CD49a+ CD107a+ TNFα- IFNγ+ NK cells and the non-responsive CD49a+ CD107a- TNFα- IFNγ- NK cell population following infection, suggesting both pathogenic and inflammatory changes in the NK cell functional profile. Our data also identified significant global differences in polyfunctionality between CD49a+ NK cells in the naïve and chronic (SHIV+) cohorts. Our work provides the first characterization of CD49a+ NK cells in tissues from RM. The significant similarities between CD49a+ NK cells from RM and what is reported from human samples justifies the importance of studying CD49a+ NK cells in this species to support preclinical animal model research.

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.

CD49a regulates the function of human decidual natural killer cells.

PROBLEM: The function of CD49a on human decidual natural killer (dNK) cells is unknown. METHOD OF STUDY: The expression of CD49a on dNK cells from human patients with recurrent spontaneous abortions or age-matched healthy controls was analyzed by flow cytometry. DNK cells were treated with CD49a neutralizing antibody and analyzed for function (cytokines production and cytotoxic activity). Long non-coding RNA (lncRNA) microarray analysis was used to identify a potential regulator of CD49a. RESULTS: DNK cells from human patients who underwent recurrent spontaneous abortions had lower levels of CD49a and increased perforin, granzyme B, and IFN-r expression, when compared to dNK cells from age-matched healthy controls. Perforin, granzyme B, and IFN-r expression levels in dNK cells were upregulated, while the migration and adhesion of dNK cells were downregulated by CD49a-neutralizing antibody. By the 51 Cr release assay, the killing activity of dNK cells also increased with CD49a neutralizing antibody. Further, lnc-49a, a newly identified lncRNA, was shown to be a positive regulator of CD49a in primary human NK cells. CONCLUSION: CD49a is involved in the regulation of dNK cells functions, including cytotoxic activity, migration, and adhesion. Further, lnc-49a is a positive regulator of CD49a in human primary dNK cells.

Cytokine-Based Generation of CD49a+Eomes-/+ Natural Killer Cell Subsets.

Recent studies have identified CD49a+Eomes- and CD49a+Eomes+ subsets of tissue-resident NK (trNK) cells in different organs of the mouse. However, the characteristics of CD49a+Eomes-/+ NK cell development and the regulation of Eomes expression in NK cells remain unclear. Here, we established an in vitro cytokine-based feeder-free system in which bone marrow progenitor cells differentiate into CD49a+ NK cells. IL-15 was identified as being the key cytokine in this system that supported the development and maintenance of CD49a+ NK cells. The CD49a+ NK cells generated were Eomes-CD49b- and shared the same phenotype as hepatic trNK cells. IL-4 induced the expression of Eomes in generated NK cells and converted them into CD49a+Eomes+ cells, which were phenotypically and functionally similar to uterine trNK cells. Moreover, the IL-4/STAT6 axis was identified as being important in the generation of CD49a+Eomes+ induced NK cells. Collectively, these studies describe an approach to generate CD49a+Eomes-/+ subsets of NK cells and demonstrate important roles for IL-15 and IL-4 in the differentiation of these cells. These findings have potential for developmental research underlying the generation of different subsets of NK cells and the application of adoptive NK cell transfer therapies.

mTOR inhibition reduces growth and adhesion of hepatocellular carcinoma cells in vitro.

Mechanistic target of rapamycin (mTOR) signaling is typically increased in hepatocellular carcinoma (HCC). A panel of HCC cell lines (HepG2, Hep3B and HuH6) was exposed to various concentrations of the mTOR inhibitors, everolimus and temsirolimus, in order to investigate their effects on cell growth, clonal formation, cell cycle progression, and adhesion and chemotactic migration using MTT and clonal cell growth assays, fluorometric detection of cell cycle phases and a Boyden chamber assay. In addition, integrin α and ß adhesion receptors were analyzed by flow cytometry and blocking studies using function blocking monoclonal antibodies were conducted to explore functional relevance. The results demonstrated that everolimus and temsirolimus significantly suppressed HCC cell growth and clonal formation, at 0.1 or 1 nM (depending on the cell line). In addition, the number of cells in G0/G1 phase was increased in response to drug treatment, whereas the number of G2/M phase cells was decreased. Drug treatment also considerably suppressed HCC cell adhesion to immobilized collagen. Integrin profiling revealed strong expression of integrin α1, α2, α6 and ß1 subtypes; and integrin α1 was upregulated in response to mTOR inhibition. Suppression of integrin α1 did not affect cell growth; however, it did significantly decrease adhesion and chemotaxis, with the influence on adhesion being greater than that on motility. Due to a positive association between integrin α1 expression and the extent of adhesion, whereby reduced receptor expression was correlated to decreased cell adhesion, it may be hypothesized that the adhesion­blocking effects of mTOR inhibitors are not associated with mechanical contact inhibition of the α1 receptor but with integrin α1­dependent suppression of oncogenic signaling, thus preventing tumor cell­matrix interaction.

CD49a Expression Defines Tissue-Resident CD8+ T Cells Poised for Cytotoxic Function in Human Skin.

Tissue-resident memory T (Trm) cells form a heterogeneous population that provides localized protection against pathogens. Here, we identify CD49a as a marker that differentiates CD8+ Trm cells on a compartmental and functional basis. In human skin epithelia, CD8+CD49a+ Trm cells produced interferon-γ, whereas CD8+CD49a- Trm cells produced interleukin-17 (IL-17). In addition, CD8+CD49a+ Trm cells from healthy skin rapidly induced the expression of the effector molecules perforin and granzyme B when stimulated with IL-15, thereby promoting a strong cytotoxic response. In skin from patients with vitiligo, where melanocytes are eradicated locally, CD8+CD49a+ Trm cells that constitutively expressed perforin and granzyme B accumulated both in the epidermis and dermis. Conversely, CD8+CD49a- Trm cells from psoriasis lesions predominantly generated IL-17 responses that promote local inflammation in this skin disease. Overall, CD49a expression delineates CD8+ Trm cell specialization in human epithelial barriers and correlates with the effector cell balance found in distinct inflammatory skin diseases.

Tissue-resident natural killer cells in the livers.

Nature killer (NK) cells are important lymphocytes of the innate immune system, well known for their pivotal roles in immune surveillance and defense against tumor and virus-infected cells. Current studies have revealed that NK cells are not a homogeneous population, but instead consist of distinct subsets with diverse characteristics. As an organ with predominant innate immunity, the liver is enriched with NK cells, among which two distinct NK cell subsets have recently been identified: conventional NK (cNK) cells and tissue-resident NK (trNK) cells. Liver trNK cells are markedly different from cNK cells in many aspects, representing a new lineage of innate lymphoid cell (ILC) family. Here, we summarize the phenotypic and functional features of liver trNK cells, and review current knowledge regarding developmental pathway of liver trNK cells. We also overview recent advances in human liver trNK cells and discuss the striking shared hallmarks of trNK cells in different tissues.

Langerhans Cells Suppress CD49a+ NK Cell-Mediated Skin Inflammation.

Recruitment of innate immune effector cells into sites of infection is a critical component of resistance to pathogen infection. Using a model of intradermal footpad injection of Candida albicans, we observed that inflammation as measured by footpad thickness and neutrophil recruitment occurred independent of adoptive immunity but was significantly reduced in MyD88(-/-) and IL-6(-/-) mice. Unexpectedly, huLangerin-DTA mice (ΔLC) that lack Langerhans cells (LC) developed increased skin inflammation and expressed higher amounts of IL-6, suggesting a suppressive role for LC. Increased inflammation also occurred in Rag1(-/-) ΔLC mice but was reversed by Ab-mediated ablation of NK cells. CXCR6(+)CD49a(+) NK cells are a liver-resident subset that can mediate inflammatory skin responses. We found that exaggerated skin inflammation was absent in ΔLC × CXCR6(-/-) mice. Moreover, the exaggerated response in ΔLC mice could be adoptively transferred with liver CD49a(+) NK cells. Finally, CD49a(+) NK cells in ΔLC but not control mice were recruited to the skin, and inhibition of their recruitment prevented the exaggerated response. Thus, in the absence of LC, CD49a(+) liver NK cells display an inappropriately proinflammatory phenotype that results in increased local skin inflammation. These data reveal a novel function for LC in the regulation of this recently described subset of skin tropic NK cells.

Toll-like receptor agonist imiquimod facilitates antigen-specific CD8+ T-cell accumulation in the genital tract leading to tumor control through IFNγ.

PURPOSE: Imiquimod is a Toll-like receptor 7 agonist used topically to treat external genital warts and basal cell carcinoma. We examined the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic human papillomavirus (HPV) vaccine comprised of a naked DNA vector expressing calreticulin fused to HPV16 E7. EXPERIMENTAL DESIGN: Using an orthotopic HPV16 E6/E7(+) syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we assessed if combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod could result in synergistic activities promoting immune-mediated tumor clearance. RESULTS: Imiquimod induced cervicovaginal accumulation of activated E7-specific CD8(+) T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8(+) T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract, which are produced in response to IFNγ receptor signaling and attract cells expressing their ligand, CXCR3. The T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as CD49a, an integrin involved in homing and retention of CD8(+) T cells at mucosal sites. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3(+) CD8(+) T cells to the genital tract. CONCLUSIONS: Several therapeutic HPV vaccination clinical trials using a spectrum of DNA vaccines, including vaccination in concert with cervical imiquimod, are ongoing. Our study identifies a mechanism by which these strategies could provide therapeutic benefit. Our findings support accumulating evidence that manipulation of the tumor microenvironment can enhance the therapeutic efficacy of strategies that induce tumor-specific T cells.

Role of integrin α1 subunits in gastric cancer patients with peritoneal dissemination.

The interaction between gastric cancer (GC) cells and the peritoneum is a critical event in peritoneal dissemination. The molecular mechanisms of this dissemination, however, remain unclear. Integrins are heterodimeric cell adhesion molecules consisting of α and ß subunits that serve as adhesion receptors for extracellular matrix proteins and cellular ligands, and may participate in GC peritoneal dissemination. In this study, we isolated fresh GC cells from a patient with peritoneal metastasis and examined them for integrin expression and investigated the role of integrin α1 subunit molecules in GC. Five clones (KGC1C2, KGC1F3, KGC1H3, KGC1E8, KGC1G10) were established from the clinical GC sample and used in an in vitro adhesion model using a single cell culture method. Each clone was transplanted into the peritoneal cavity of SCID mice, where each clone formed tumors and caused conglutination of organs in the abdominal cavity. We analyzed the expression of integrin subunits for each clone by flow cytometry and found that the expression ratio of α1 subunits paired with ß1 subunits was detected at higher levels than other subunits. To verify that anti-integrin α1 subunit (CD49a) antibody inhibits cell adhesion in an in vitro adhesion assay, each clone was treated with anti-CD49a antibody, which significantly inhibited cell adhesion compared to the untreated group. Characterization of α1 subunit expression in GC may be useful in optimizing treatments for different individuals. Having high metastatic abilities, these 5 new GC clones may be beneficial for analyzing integrin function in tumor metastasis.

Key role of alpha(1)beta(1)-integrin in the activation of PI3-kinase-Akt by flow (shear stress) in resistance arteries.

Resistance arteries are the site of the earliest manifestations of many cardiovascular and metabolic diseases. Flow (shear stress) is the main physiological stimulus for the endothelium through the activation of vasodilatory pathways generating flow-mediated dilation (FMD). The role of FMD in local blood flow control and angiogenesis is well established, and alterations in FMD are early markers of cardiovascular disorders. alpha(1)-Integrin, which has a role in angiogenesis, could be involved in FMD. FMD was studied in mesenteric resistance arteries (MRA) isolated in arteriographs. The role of alpha(1)-integrins in FMD was tested with selective antibodies and mice lacking the gene encoding for alpha(1)-integrins. Both anti-alpha(1) blocking antibodies and genetic deficiency in alpha(1)-integrin in mice (alpha(1)(-/-)) inhibited FMD without affecting receptor-mediated (acetylcholine) endothelium-dependent dilation or endothelium-independent dilation (sodium nitroprusside). Similarly, vasoconstrictor tone (myogenic tone and phenylephrine-induced contraction) was not affected. In MRA phosphorylated Akt and phosphatidylinositol 3-kinase (PI3-kinase) were significantly lower in alpha(1)(-/-) mice than in alpha(1)(+/+) mice, although total Akt and endothelial nitric oxide synthase (eNOS) were not affected. Pharmacological blockade of PI3-kinase-Akt pathway with LY-294002 inhibited FMD. This inhibitory effect of LY-294002 was significantly lower in alpha(1)(-/-) mice than in alpha(1)(+/+) mice. Thus alpha(1)-integrin has a key role in flow (shear stress)-dependent vasodilation in resistance arteries by transmitting the signal to eNOS through activation of PI3-kinase and Akt. Because of the central role of flow (shear stress) activation of the endothelium in vascular disorders, this finding opens new perspectives in the pathophysiology of the microcirculation and provides new therapeutic targets.

Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype.

Human bone marrow mesenchymal stem cells (MSC) generate, via a fibroblast colony-forming unit (CFU-F), osteo-chondroblastic cells as well as adipocytes and stromacytes. To date, these stem cells are isolated indirectly using a cell culture method and phenotyped as CD45 negative while the in vivo counterparts are undetermined. Our aim was to develop a direct selection method and to determine the phenotype of the MSC isolated in this way. Mesenchymal cells were selected with anti-CD49a and/or anti-CD45 antibodies using either flow cytometry or a magnetic beads method. All CFU-F were always detected in the small population of CD49a-positive cells. These CFU retained their differentiation potential and gave rise to osteo-chondroblastic cells, adipocytes and stromacytes. Phenotypic studies on uncultured cells revealed a CD45med,low, CD34low, HLA-II- cell population. Flow cytometry cell sorting showed that MSC with CFU-F potential were obtained only from a CD49a+/CD45med,low population. In addition, when cultured, they clearly became CD45-, CD34-, HLA-II-, CD49a+. These results confirmed that MSC can be directly selected easily from human bone marrow using magnetic beads without altering their differentiation potential. These cells expressed mildly the haematopoietic marker CD45, which was dramatically downregulated by in vitro culture. The expression of CD45 coupled to CD49a thus enabled direct selection of the MSC.

Analysis of cell-free human alpha1 integrin with a monoclonal antibody to the I-domain: detection in ocular fluid and function as an adhesion substrate.

The alpha1 beta1 integrin, an inserted (1) domain containing collagen receptor, is expressed in the cell surface membrane of normal and malignant cells, and may play a role in their migration through tissues or in metastatic spread. Here we report that a functional anti-human alpha1beta1 integrin monoclonal antibody (mAb) (1B3.1) directly and specifically binds plastic bound recombinant human alpha1 I-domain protein containing the collagen binding site. Detection was diminished by acidification of the I-domain protein but was enhanced by increasing concentrations of Mg2+ cation. Furthermore, we detected binding of the mAb to proteins from the ocular fluids of 6 patients, with the highest concentration, corresponding to 22.1 ng/ml of I-domain, found in a sample from the eye of a patient with metastatic lung adenocarcinoma. Interestingly, we found that both SKNSH neuroblastoma cells and virally transformed human T cells adhered specifically to plastic wells coated with either immobilized collagen IV or alpha1 I-domain. MAb I B3.1 inhibited adhesion to collagen IV but not to immobilized I-domain. These results suggest a novel function for cell free alpha1 I-domain as a substrate for cellular adhesion, which may have relevance in tumor spread in vivo.