NKG2A Blockade Potentiates CD8 T Cell Immunity Induced by Cancer Vaccines.

Tumor-infiltrating CD8 T cells were found to frequently express the inhibitory receptor NKG2A, particularly in immune-reactive environments and after therapeutic cancer vaccination. High-dimensional cluster analysis demonstrated that NKG2A marks a unique immune effector subset preferentially co-expressing the tissue-resident CD103 molecule, but not immune checkpoint inhibitors. To examine whether NKG2A represented an adaptive resistance mechanism to cancer vaccination, we blocked the receptor with an antibody and knocked out its ligand Qa-1b, the conserved ortholog of HLA-E, in four mouse tumor models. The impact of therapeutic vaccines was greatly potentiated by disruption of the NKG2A/Qa-1b axis even in a PD-1 refractory mouse model. NKG2A blockade therapy operated through CD8 T cells, but not NK cells. These findings indicate that NKG2A-blocking antibodies might improve clinical responses to therapeutic cancer vaccines.

Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation.

Costimulation is required for optimal T cell activation, yet it is unclear whether poxviruses dedicatedly subvert costimulation during infection. Here, we report that the secreted M2 protein encoded by cowpox virus (CPXV) specifically interacts with human and murine B7.1 (CD80) and B7.2 (CD86). We also show that M2 competes with CD28 and CTLA4 for binding to cell surface B7 ligands, with stronger efficacy against CD28. Functionally, recombinant M2 and culture supernatants from wild-type (WT) but not M2-deficient (∆M2) CPXV-infected cells can potently suppress B7 ligand-mediated T cell proliferation and interleukin-2 (IL-2) production. Furthermore, we observed increased antiviral CD4 and CD8 T cell responses in C57BL/6 mice challenged by ∆M2 CPXV compared with WT virus. These differences in immune responses to ∆M2 and WT CPXV were not observed in CD28-deficient mice. Taken together, our findings define a mechanism of viral sabotage of T cell activation that highlights the role of CD28 costimulation in host defense against poxvirus infections.

Activation Receptor-Dependent IFN-γ Production by NK Cells Is Controlled by Transcription, Translation, and the Proteasome.

NK cells can recognize target cells such as virus-infected and tumor cells through integration of activation and inhibitory receptors. Recognition by NK cells can lead to direct lysis of the target cell and production of the signature cytokine IFN-γ. However, it is unclear whether stimulation through activation receptors alone is sufficient for IFN-γ production. In this study, we show that NK activation receptor engagement requires additional signals for optimal IFN-γ production, which could be provided by IFN-ß or IL-12. Stimulation of murine NK cells with soluble Abs directed against NK1.1, Ly49H, Ly49D, or NKp46 required additional stimulation with cytokines, indicating that a range of activation receptors with distinct adaptor molecules require additional stimulation for IFN-γ production. The requirement for multiple signals extends to stimulation with primary m157-transgenic target cells, which triggers the activation receptor Ly49H, suggesting that NK cells do require multiple signals for IFN-γ production in the context of target cell recognition. Using quantitative PCR and RNA flow cytometry, we found that cytokines, not activating ligands, act on NK cells to express Ifng transcripts. Ly49H engagement is required for IFN-γ translational initiation. Results using inhibitors suggest that the proteasome-ubiquitin-IKK-TPL2-MNK1 axis was required during activation receptor engagement. Thus, this study indicates that activation receptor-dependent IFN-γ production is regulated on the transcriptional and translational levels.

Cutting Edge: Human CD49e- NK Cells Are Tissue Resident in the Liver.

Most knowledge on NK cells is based on studies of what are now known as conventional NK cells in the mouse spleen or human peripheral blood. However, recent studies in mice indicate the presence of tissue-resident NK cells in certain organs, such as the liver, that display different markers and transcription factor dependencies as compared with conventional NK cells. In this study, we provide evidence from cytometry by time-of-flight analysis and humanized mice indicating that human CD49e- NK cells are tissue resident in the liver. Thus, these studies indicate that tissue-resident NK cells are evolutionarily conserved in humans and mice, providing a foundation to explore their role in human disease.

Dual Requirement of Cytokine and Activation Receptor Triggering for Cytotoxic Control of Murine Cytomegalovirus by NK Cells.

Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

Immune Evasion by Epstein-Barr Virus.

Epstein-Bar virus (EBV) is widespread within the human population with over 90% of adults being infected. In response to primary EBV infection, the host mounts an antiviral immune response comprising both innate and adaptive effector functions. Although the immune system can control EBV infection to a large extent, the virus is not cleared. Instead, EBV establishes a latent infection in B lymphocytes characterized by limited viral gene expression. For the production of new viral progeny, EBV reactivates from these latently infected cells. During the productive phase of infection, a repertoire of over 80 EBV gene products is expressed, presenting a vast number of viral antigens to the primed immune system. In particular the EBV-specific CD4+ and CD8+ memory T lymphocytes can respond within hours, potentially destroying the virus-producing cells before viral replication is completed and viral particles have been released. Preceding the adaptive immune response, potent innate immune mechanisms provide a first line of defense during primary and recurrent infections. In spite of this broad range of antiviral immune effector mechanisms, EBV persists for life and continues to replicate. Studies performed over the past decades have revealed a wide array of viral gene products interfering with both innate and adaptive immunity. These include EBV-encoded proteins as well as small noncoding RNAs with immune-evasive properties. The current review presents an overview of the evasion strategies that are employed by EBV to facilitate immune escape during latency and productive infection. These evasion mechanisms may also compromise the elimination of EBV-transformed cells, and thus contribute to malignancies associated with EBV infection.

Tissue-specific features of innate lymphoid cells in antiviral defense.

Innate lymphocytes (ILCs) rapidly respond to and protect against invading pathogens and cancer. ILCs include natural killer (NK) cells, ILC1s, ILC2s, ILC3s, and lymphoid tissue inducer (LTi) cells and include type I, type II, and type III immune cells. While NK cells have been well recognized for their role in antiviral immunity, other ILC subtypes are emerging as players in antiviral defense. Each ILC subset has specialized functions that uniquely impact the antiviral immunity and health of the host depending on the tissue microenvironment. This review focuses on the specialized functions of each ILC subtype and their roles in antiviral immune responses across tissues. Several viruses within infection-prone tissues will be highlighted to provide an overview of the extent of the ILC immunity within tissues and emphasize common versus virus-specific responses.

Expression of a single inhibitory Ly49 receptor is sufficient to license NK cells for effector functions.

Natural killer (NK) cells recognize target cells through germline-encoded activation and inhibitory receptors enabling effective immunity against viruses and cancer. The Ly49 receptor family in the mouse and killer immunoglobin-like receptor family in humans play a central role in NK cell immunity through recognition of MHC class I and related molecules. Functionally, these receptor families are involved in licensing and rejection of MHC-I-deficient cells through missing-self. The Ly49 family is highly polymorphic, making it challenging to detail the contributions of individual Ly49 receptors to NK cell function. Herein, we showed mice lacking expression of all Ly49s were unable to reject missing-self target cells in vivo, were defective in NK cell licensing, and displayed lower KLRG1 on the surface of NK cells. Expression of Ly49A alone on a H-2Dd background restored missing-self target cell rejection, NK cell licensing, and NK cell KLRG1 expression. Thus, a single inhibitory Ly49 receptor is sufficient to license NK cells and mediate missing-self in vivo.

Cis-regulatory evolution of the recently expanded Ly49 gene family.

Comparative genomics has revealed the rapid expansion of multiple gene families involved in immunity. Members within each gene family often evolved distinct roles in immunity. However, less is known about the evolution of their epigenome and cis-regulation. Here we systematically profile the epigenome of the recently expanded murine Ly49 gene family that mainly encode either inhibitory or activating surface receptors on natural killer cells. We identify a set of cis-regulatory elements (CREs) for activating Ly49 genes. In addition, we show that in mice, inhibitory and activating Ly49 genes are regulated by two separate sets of proximal CREs, likely resulting from lineage-specific losses of CRE activity. Furthermore, we find that some Ly49 genes are cross-regulated by the CREs of other Ly49 genes, suggesting that the Ly49 family has begun to evolve a concerted cis-regulatory mechanism. Collectively, we demonstrate the different modes of cis-regulatory evolution for a rapidly expanding gene family.

Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses.

One half of a group of 20 patients with human papillomavirus type 16 (HPV16)-induced vulvar intraepithelial neoplasia grade 3 displayed a complete regression (CR) after therapeutic vaccination with HPV16 E6/E7 synthetic long peptides. Patients with relatively larger lesions generally did not display a CR. To investigate immune correlates of treatment failure, patients were grouped according to median lesion size at study entry, and HPV16-specific immunity was analyzed at different time points by complementary immunological assays. The group of patients with smaller lesions displayed stronger and broader vaccine-prompted HPV16-specific proliferative responses with higher IFNgamma (P = 0.0003) and IL-5 (P < 0.0001) levels than patients with large lesions. Characteristically, this response was accompanied by a distinct peak in cytokine levels after the first vaccination. In contrast, the patient group with larger lesions mounted higher frequencies of HPV16-specific CD4(+)CD25(+)Foxp3(+) T cells (P = 0.005) and displayed a lower HPV16-specific IFNgamma/IL-10 ratio after vaccination (P < 0.01). No disparity in T memory immunity to control antigens was found, indicating that the differences in HPV-specific immunity did not reflect general immune failure. We observed a strong correlation between a defined set of vaccine-prompted specific immune responses and the clinical efficacy of therapeutic vaccination. Notably, a high ratio of HPV16-specific vaccine-prompted effector T cells to HPV16-specific CD4(+)CD25(+)Foxp3(+) T cells was predictive of clinical success. Foxp3(+) T cells have been associated previously with impaired immunity in malignancies. Here we demonstrate that the vaccine-prompted level of this population is associated with early treatment failure.

NK cell expansion requires HuR and mediates control of solid tumors and long-term virus infection.

Natural killer (NK) cells are lymphocytes capable of controlling tumors and virus infections through direct lysis and cytokine production. While both T and NK cells expand and accumulate in affected tissues, the role of NK cell expansion in tumor and viral control is not well understood. Here, we show that posttranscriptional regulation by the RNA-binding protein HuR is essential for NK cell expansion without negatively affecting effector functions. HuR-deficient NK cells displayed defects in the metaphase of the cell cycle, including decreased expression and alternative splicing of Ska2, a component of the spindle and kinetochore complex. HuR-dependent NK cell expansion contributed to long-term cytomegalovirus control and facilitated control of subcutaneous tumors but not tumor metastases in two independent tumor models. These results show that posttranscriptional regulation by HuR specifically affects NK cell expansion, which is required for the control of long-term virus infection and solid tumors, but not acute infection or tumor metastases, highlighting fundamental differences with antigen-specific T cell control.

Natural killer cell effector functions in antiviral defense.

Natural killer (NK) cells are innate lymphoid cells involved in the control of tumors and viral infections. They provide protection by producing cytokines and by directly lysing target cells. Both effector mechanisms have been identified to contribute to viral control, depending on the context of infection. Activation of NK cells depends on the integration of signals received by cytokine receptors and activation and inhibitory receptors recognizing ligands expressed by virus-infected cells. While the control of viral infections by NK cells is well established, the signals perceived by NK cells and how these signals integrate to mediate optimal viral control have been focus of ongoing research. Here, we discuss the current knowledge on NK cell activation and integration of signals that lead to interferon gamma production and cytotoxicity in viral infections. We review NK cell interactions with viruses, with particular focus on murine cytomegalovirus studies, which helped elucidate crucial aspects of antiviral NK cell immunity.

The transcription factor Bach2 negatively regulates murine natural killer cell maturation and function.

BTB domain And CNC Homolog 2 (Bach2) is a transcription repressor that actively participates in T and B lymphocyte development, but it is unknown if Bach2 is also involved in the development of innate immune cells, such as natural killer (NK) cells. Here, we followed the expression of Bach2 during murine NK cell development, finding that it peaked in immature CD27+CD11b+ cells and decreased upon further maturation. Bach2 showed an organ and tissue-specific expression pattern in NK cells. Bach2 expression positively correlated with the expression of transcription factor TCF1 and negatively correlated with genes encoding NK effector molecules and those involved in the cell cycle. Lack of Bach2 expression caused changes in chromatin accessibility of corresponding genes. In the end, Bach2 deficiency resulted in increased proportions of terminally differentiated NK cells with increased production of granzymes and cytokines. NK cell-mediated control of tumor metastasis was also augmented in the absence of Bach2. Therefore, Bach2 is a key checkpoint protein regulating NK terminal maturation.

Influenza matrix 1-specific human CD4+ FOXP3+ and FOXP3(-) regulatory T cells can be detected long after viral clearance.

Control and termination of infection with Influenza A virus is associated with increased IL-10 production in mouse models. Notably, IL-10 can be produced by Treg. Therefore, we investigated whether the population of IL-10-producing influenza-specific CD4(+) T cells comprised Treg as they are potent suppressors of the adaptive immune response. Influenza-specific IL-10-producing T cells were detected in all human donors displaying influenza-specific immunity. Isolation of Matrix 1 protein-specific IL-10-producing T-cell clones revealed that a substantial proportion of these T-cell clones displayed the capacity to suppress effector cells, functionally identifying them as Treg. Both FOXP3(+) and FOXP3(-) CD4(+) Treg were isolated and all were able to exert their suppressive capacity when stimulated with cognate antigen, including influenza virus-infected cells. In vitro suppression was not mediated by IL-10 but involved interference with the IL-2 axis. The isolated Treg suppressed amongst others the IL-2 production of influenza-specific T-helper cells as well as partially prevented the upregulation of the high-affinity IL-2 receptor on CD8 effector cells. So far the induction of virus-specific Treg has only been studied in the context of chronic viral infections. This study demonstrates that virus-specific Treg can also be induced by viruses that are rapidly cleared in humans.

HIF1α is required for NK cell metabolic adaptation during virus infection.

Natural killer (NK) cells are essential for early protection against virus infection and must metabolically adapt to the energy demands of activation. Here, we found upregulation of the metabolic adaptor hypoxia-inducible factor-1α (HIF1α) is a feature of mouse NK cells during murine cytomegalovirus (MCMV) infection in vivo. HIF1α-deficient NK cells failed to control viral load, causing increased morbidity. No defects were found in effector functions of HIF1αKO NK cells; however, their numbers were significantly reduced. Loss of HIF1α did not affect NK cell proliferation during in vivo infection and in vitro cytokine stimulation. Instead, we found that HIF1α-deficient NK cells showed increased expression of the pro-apoptotic protein Bim and glucose metabolism was impaired during cytokine stimulation in vitro. Similarly, during MCMV infection HIF1α-deficient NK cells upregulated Bim and had increased caspase activity. Thus, NK cells require HIF1α-dependent metabolic functions to repress Bim expression and sustain cell numbers for an optimal virus response.

Virus infection is controlled by hematopoietic and stromal cell sensing of murine cytomegalovirus through STING.

Recognition of DNA viruses, such as cytomegaloviruses (CMVs), through pattern-recognition receptor (PRR) pathways involving MyD88 or STING constitute a first-line defense against infections mainly through production of type I interferon (IFN-I). However, the role of these pathways in different tissues is incompletely understood, an issue particularly relevant to the CMVs which have broad tissue tropisms. Herein, we contrasted anti-viral effects of MyD88 versus STING in distinct cell types that are infected with murine CMV (MCMV). Bone marrow chimeras revealed STING-mediated MCMV control in hematological cells, similar to MyD88. However, unlike MyD88, STING also contributed to viral control in non-hematological, stromal cells. Infected splenic stromal cells produced IFN-I in a cGAS-STING-dependent and MyD88-independent manner, while we confirmed plasmacytoid dendritic cell IFN-I had inverse requirements. MCMV-induced natural killer cytotoxicity was dependent on MyD88 and STING. Thus, MyD88 and STING contribute to MCMV control in distinct cell types that initiate downstream immune responses.

Control of Viral Infection by Natural Killer Cell Inhibitory Receptors.

Major histocompatibility complex class I (MHC-I)-restricted immune responses are largely attributed to cytotoxic T lymphocytes (CTLs). However, natural killer (NK) cells, as predicted by the missing-self hypothesis, have opposing requirements for MHC-I, suggesting that they may also demonstrate MHC-I-restricted effects. In mice, the Ly49 inhibitory receptors prevent NK cell killing of missing-self targets in effector responses, and they have a proposed second function in licensing or educating NK cells via self-MHC-I in vivo. Here we show MHC-I-restricted control of murine cytomegalovirus (MCMV) infection in vivo that is NK cell dependent. Using mice lacking specific Ly49 receptors, we show that control of MCMV requires inhibitory Ly49 receptors and an inhibitory signaling motif and the capacity for MCMV to downregulate MHC-I. Taken together, these data provide definitive evidence that the inhibitory receptors are required for missing-self rejection and are relevant to MHC-I-restricted NK cell control of a viral infection in vivo.

Surgery followed by persistence of high-grade squamous intraepithelial lesions is associated with the induction of a dysfunctional HPV16-specific T-cell response.

PURPOSE: To characterize HPV16 E6- and E7-specific T-cell immunity in patients with high-grade squamous intraepithelial lesions (HSIL). EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells isolated from 38 patients with HPV16+ HSIL were used to determine the magnitude, breadth, and polarization of HPV16-specific T-cell responses by proliferation assays and cytokine assays. Furthermore, HSIL-infiltrating T cells isolated from 7 cases were analyzed for the presence of HPV16 E6- and/or E7-specific T cells, phenotyped, and tested for the specific production of IFN-gamma and interleukin-10 as well as for their capacity to suppress immune responses. RESULTS: HPV16-specific T-cell responses were absent in the circulation of the majority (approximately 60%) of patients who visit the clinic for treatment of a HPV16+ HSIL lesion. Notably, HPV16-specific T-cell reactivity was predominantly detected in patients returning to the clinic for repetitive treatment of a persistent or recurrent HPV16+ HSIL lesion after initial destructive treatment. The majority (> 70%) of these HPV16-specific T-cell responses did not secrete proinflammatory cytokines, indicating that most of the subjects, although in principle able to mount a HPV16-specific immune response, fail to develop protective cellular immunity. This notion is sustained by our observation that only three HSIL-infiltrating T-cell cultures contained HPV16-specific T cells, one of which clearly consisted of HPV16 E7-specific regulatory T cells. CONCLUSIONS: The presence of HPV16-specific T cells with a non-Th1/Th2 cytokine and even suppressive signature in patients with HSIL may affect the outcome of vaccine approaches aiming at reinforcing human papillomavirus-specific immunity to attack human papillomavirus-induced lesions.

Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine.

PURPOSE: The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides vaccine on the antigen-specific T-cell response in cervical cancer patients. EXPERIMENTAL DESIGN: Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. HPV16-specific T-cell immune responses were analyzed by evaluating the magnitude, breadth, type, and polarization by proliferation assays, IFN gamma-ELISPOT, and cytokine production and phenotyped by the T-cell markers CD4, CD8, CD25, and Foxp3. RESULTS: Vaccine-induced T-cell responses against HPV16 E6 and E7 were detected in six of six and five of six patients, respectively. These responses were broad, involved both CD4(+) and CD8(+) T cells, and could be detected up to 12 months after the last vaccination. The vaccine-induced responses were dominated by effector type CD4(+)CD25(+)Foxp3(-) type 1 cytokine IFN gamma-producing T cells but also included the expansion of T cells with a CD4(+)CD25(+)Foxp3(+) phenotype. CONCLUSIONS: The HPV16 E6 and E7 synthetic long peptides vaccine is highly immunogenic, in that it increases the number and activity of HPV16-specific CD4(+) and CD8(+) T cells to a broad array of epitopes in all patients. The expansion of CD4(+) and CD8(+) tumor-specific T cells, both considered to be important in the antitumor response, indicates the immunotherapeutic potential of this vaccine. Notably, part of the vaccine-induced T cells display a CD4(+)CD25(+)Foxp3(+) phenotype that is frequently associated with regulatory T-cell function, suggesting that strategies to disarm this subset of T cells should be considered as components of immunotherapeutic modalities against HPV-induced cancers.

High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer.

In a prospective study, we have examined the tumor-specific immune response in a group of 59 patients with human papillomavirus (HPV) 16-positive (HPV16(+))-induced or HPV18(+)-induced cervical cancer. Local antitumor immunity was analyzed by the enumeration of tumor-infiltrating dendritic cells and CD4+, CD8+, and regulatory T cells as well as by calculation of the ratio of CD8+/CD4+ T cells and CD8+/regulatory T cells. Systemic tumor-specific immunity was assessed by determination of the HPV E6- and/or E7-specific T-cell response in the blood of these patients. Finally, these variables were evaluated with respect to known histopathologic prognostic variables, including the absence (LN-) or presence (LN+) of lymph node metastases. Stratification according to the lymph node status of patients revealed a significantly stronger CD8+ T-cell tumor infiltration, a higher CD8+/CD4+ T-cell ratio, and higher CD8+/regulatory T-cell ratio in the group of patients in which the tumor failed to metastasize to the tumor-draining lymph node. Subdivision according to the presence (IR+) or absence (IR-) of circulating HPV-specific T cells disclosed that the highest number of tumor-infiltrating CD8+ T cells was found in the group of LN- patients displaying a concomitant systemic tumor-specific immune response (LN-IR+). CD8+ T-cell infiltration in LN-IR- patients was comparable with that of LN+ patients. In cervical cancer, the absence of lymph node metastases is strongly associated with a better prognosis. Our data indicate that, especially in a subgroup of LN- patients, a strong and effective interaction between immune system and tumor exists. This subgroup of cervical cancer patients may have the best prognosis.