Tissue Determinants of Human NK Cell Development, Function, and Residence.

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT.

Mass cytometry reveals single-cell kinetics of cytotoxic lymphocyte evolution in CMV-infected renal transplant patients.

Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8+ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C+CD57+FcεRIγ-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C+CD57+FcεRIγlow-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C+CD8+ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.

Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy.

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2 + influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca + + influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2 + flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70. Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells.

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.

CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset.

Natural killer (NK) cells are innate immune lymphocytes that express a heterogeneous repertoire of germline-encoded receptors and undergo a distinct pattern of maturation. CD57 is a marker of terminal differentiation on human CD8(+) T cells. Very few newborn or fetal NK cells express CD57; however, the frequency of CD57-bearing NK cells increases with age. We assessed the transcriptional, phenotypic, and functional differences between CD57(+) and CD57(-) NK cells within the CD56(dim) mature NK subset. CD57(+) NK cells express a repertoire of NK-cell receptors, suggestive of a more mature phenotype, and proliferate less when stimulated with target cells and/or cytokines. By contrast, a higher frequency of CD57(+) NK cells produced interferon-γ and demonstrated more potent lytic activity when these cells were stimulated through the activating receptor CD16; however, they are less responsive to stimulation by interleukin-12 and interleukin-18. Finally, CD57 expression is induced on CD57(-)CD56(dim) NK cells after activation by interleukin-2. A combination of a mature phenotype, a higher cytotoxic capacity, a higher sensitivity to stimulation via CD16, with a decreased responsiveness to cytokines, and a decreased capacity to proliferate suggest that CD57(+) NK cells are highly mature and might be terminally differentiated.

Diminished cell proliferation promotes natural killer cell adaptive-like phenotype by limiting FcεRIγ expression.

Human adaptive-like natural killer (NK) cells express low levels of FcεRIγ (FcRγ-/low) and are reported to accumulate during COVID-19 infection; however, the mechanism underlying and regulating FcRγ expression in NK cells has yet to be fully defined. We observed lower FcRγ protein expression in NK cell subsets from lung transplant patients during rapamycin treatment, suggesting a link with reduced mTOR activity. Further, FcRγ-/low NK cell subsets from healthy donors displayed reduced mTOR activity. We discovered that FcRγ upregulation is dependent on cell proliferation progression mediated by IL-2, IL-15, or IL-12, is sensitive to mTOR suppression, and is inhibited by TGFß or IFNα. Accordingly, the accumulation of adaptive-like FcRγ-/low NK cells in COVID-19 patients corresponded to increased TGFß and IFNα levels and disease severity. Our results show that an adaptive-like NK cell phenotype is induced by diminished cell proliferation and has an early prognostic value for increased TGFß and IFNα levels in COVID-19 infection associated with disease severity.

NK and CD8+ T cell phenotypes predict onset and control of CMV viremia after kidney transplant.

CMV causes mostly asymptomatic but lifelong infection. Primary infection or reactivation in immunocompromised individuals can be life-threatening. CMV viremia often occurs in solid organ transplant recipients and associates with decreased graft survival and higher mortality. Furthering understanding of impaired immunity that allows CMV reactivation is critical to guiding antiviral therapy and examining the effect of CMV on solid organ transplant outcomes. This study characterized longitudinal immune responses to CMV in 31 kidney transplant recipients with CMV viremia and matched, nonviremic recipients. Recipients were sampled 3 and 12 months after transplant, with additional samples 1 week and 1 month after viremia. PBMCs were stained for NK and T cell markers. PBMC transcriptomes were characterized by RNA-Seq. Plasma proteins were quantified by Luminex. CD8+ T cell transcriptomes were characterized by single-cell RNA-Seq. Before viremia, patients had high levels of IL-15 with concurrent expansion of immature CD56bright NK cells. After viremia, mature CD56dim NK cells and CD28-CD8+ T cells upregulating inhibitory and NK-associated receptors were expanded. Memory NK cells and NK-like CD28-CD8+ T cells were associated with control of viremia. These findings suggest that signatures of innate activation may be prognostic for CMV reactivation after transplant, while CD8+ T cell functionality is critical for effective control of CMV.

Combining angiogenic gene and stem cell therapies for myocardial infarction.

BACKGROUND: Transplantation of stem cells from various sources into infarcted hearts has the potential to promote myocardial regeneration. However, the regenerative capacity is limited partly as a result of the low survival rate of the transplanted cells in the ischemic myocardium. In the present study, we tested the hypothesis that combining cell and angiogenic gene therapies would provide additive therapeutic effects via co-injection of bone marrow-derived mesenchymal stem cells (MSCs) with an adeno-associated viral vector (AAV), MLCVEGF, which expresses vascular endothelial growth factor (VEGF) in a cardiac-specific and hypoxia-inducible manner. METHODS: MSCs isolated from transgenic mice expressing green fluorescent protein and MLCVEGF packaged in AAV serotype 1 capsid were injected into mouse hearts at the border of ischemic area, immediately after occlusion of the left anterior descending coronary, individually or together. Engrafted cells were detected and quantified by real-time polymerase chain reaction and immunostaining. Angiogenesis and infarct size were analyzed on histological and immunohistochemical stained sections. Cardiac function was analyzed by echocardiography. RESULTS: We found that co-injection of AAV1-MLCVEGF with MSCs reduced cell loss. Although injection of MSCs and AAV1-MLCVEGF individually improved cardiac function and reduced infarct size, co-injection of MSC and AAV1-MLCVEGF resulted in the best improvement in cardiac function as well as the smallest infarct among all groups. Moreover, injection of AAV1-MLCVEGF induced neovasculatures. Nonetheless, injection of MSCs attracted endogenous stem cell homing and increased scar thickness. CONCLUSIONS: Co-injection of MLCVEGF and MSCs in ischemic hearts can result in better cardiac function and MSC survival, compared to their individual injections, as a result of the additive effects of each therapy.

VEGF improves survival of mesenchymal stem cells in infarcted hearts.

Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infarcted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16(INK), p21 and p19(ARF). VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs or VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI.

Additive effect of AAV-mediated angiopoietin-1 and VEGF expression on the therapy of infarcted heart.

Vascular endothelial growth factor (VEGF) is a key angiogenic factor and has been used experimentally for induction of neovasculature in ischemic myocardium. However, blood vessels induced by VEGF are immature. Angiopoietin-1 (ang-1) has the ability to recruit and sustain periendothelial support cells and promote vascular maturation. Thus, co-expression of the two may yield a better result than expression of either one alone. Two adeno-associated viral vectors (AAV), CMVVEGF and CMVang-1 with the CMV promoter driving VEGF or ang-1 gene expression, respectively, were injected into ischemic mouse hearts individually or together in different ratios. The results show that co-injected groups had more capillaries than the CMVang-1 group and similar densities of capillaries and alpha-actin positive vessels as the CMVVEGF group. Neovasculature induced by CMVVEGF was leaky. In contrast, neovasculature in CMVang-1-injected or CMVVEGF and CMVang-1 co-injected hearts was less leaky than that in CMVVEGF-injected hearts. The group that received CMVang-1 and CMVVEGF in a 1:1 ratio had the smallest infarct size and best cardiac function and regional wall movement among all the groups. We conclude that ang-1 and VEGF can compensate for each others' shortcomings and yield a better therapeutic effect by acting together.

Adeno-associated viral vector-mediated hypoxia response element-regulated gene expression in mouse ischemic heart model.

Intramyocardial injection of genes encoding angiogenic factors could provide a useful approach for the treatment of ischemic heart disease. However, uncontrolled expression of angiogenic factors in vivo may cause some unwanted side effects, such as hemangioma formation, retinopathy, and arthritis. It may also induce occult tumor growth and artherosclerotic plaque progression. Because hypoxia-inducible factor 1 is up-regulated in a variety of hypoxic conditions and it regulates gene expression by binding to a cis-acting hypoxia-responsive element (HRE), we propose to use HRE, found in the 3' end of the erythropoietin gene to control gene expression in ischemic myocardium. A concatemer of nine copies of the consensus sequence of HRE isolated from the erythropoietin enhancer was used to mediate hypoxia induction. We constructed two adeno-associated viral vectors in which LacZ and vascular endothelial growth factor (VEGF) expressions were controlled by this HRE concatemer and a minimal simian virus 40 promoter. Both LacZ and VEGF expression were induced by hypoxia and/or anoxia in several cell lines transduced with these vectors. The functions of these vectors in ischemic myocardium were tested by injecting them into normal and ischemic mouse myocardium created by occlusion of the left anterior descending coronary artery. The expression of LacZ gene was induced eight times and of VEGF 20 times in ischemic myocardium compared with normal myocardium after the viral vector transduction. Hence, HRE is a good candidate for the control of angiogenic factor gene expression in ischemic myocardium.

Adeno-associated viral vector delivers cardiac-specific and hypoxia-inducible VEGF expression in ischemic mouse hearts.

It has been shown that the adeno-associated virus (AAV) vector can deliver the VEGF gene efficiently into the ischemic mouse myocardium. However, the AAV genomes can be found in extracardiac organs after intramyocardial injection. To limit unwanted VEGF expression in organs other than the heart, we tested the use of the cardiac myosin light chain 2v (MLC-2v) promoter and the hypoxia-response element to mediate cardiac-specific and hypoxia-inducible VEGF expression. An AAV vector, MLCVEGF, with 250 bp of the MLC-2v promoter and nine copies of the hypoxia-response element driving VEGF expression, was constructed. Gene expression was studied in vitro by infection of rat cardiomyocytes, rat skeletal myocytes, and mouse fibroblasts with the vector and in vivo by direct injection of the vector into normal and ischemic mouse hearts. With MLCVEGF infection, VEGF expression was higher in cardiomyocytes than the other two cell lines and was hypoxiainducible. VEGF expression was also higher in ischemic hearts than in normal hearts. No VEGF expression was detectable in organs with detectable MLCVEGF vectors other than the heart. MLCVEGF-injected ischemic hearts had more capillaries and small vessels around the injection site, smaller infarct size, and better cardiac function than the negative controls. Hence, MLCVEGF can mediate cardiac-specific and hypoxia-inducible VEGF expression, neoangiogenesis, infarct-size reduction, and cardiac functional improvement.