The HHV-6A Proteins U20 and U21 Target NKG2D Ligands to Escape Immune Recognition

The coevolution of the human immune system and herpesviruses led to the emergence and diversification of both cellular danger molecules recognized by immune cells on the one hand and viral countermeasures that prevent the expression of these proteins on infected cells on the other. There are eight ligands for the activating receptor NKG2D in humans – MICA, MICB, ULBP1-6. Several of them are induced and surface-expressed on herpesvirus-infected cells to serve as danger signals to activate the immune system. Therefore, these ligands are frequently targeted for suppression by viral immune evasion mechanisms. Mechanisms to downregulate NKG2D ligands and thereby escape immune recognition have been identified in all other human herpesviruses (HHV), except for HHV-6A. In this study, we identify two HHV-6A encoded immunoevasins, U20 and U21, which suppress the expression of the NKG2D ligands ULBP1 and ULBP3, respectively, during infection. Additionally, MICB is targeted by a so far unexplored viral protein. Due to the diminished NKG2D ligand surface expression on infected cells, recognition of HHV-6A infected cells by innate immune cells is impaired. Importantly, our study indicates that immune escape mechanisms between the related herpesviruses HHV-6A and HHV-6B are evolutionary conserved as the same NKG2D ligands are targeted. Our data contribute an additional piece of evidence for the importance of the NKG2D receptor – NKG2D ligand axis during human herpesvirus infections and sheds light on immune evasion mechanisms of HHV-6A.

Tumor-derived NKG2D ligand sMIC reprograms NK cells to an inflammatory phenotype through CBM signalosome activation

Natural Killer (NK) cell dysfunction is associated with poorer clinical outcome in cancer patients. What regulates NK cell dysfunction in tumor microenvironment is not well understood. Here, we demonstrate that the human tumor-derived NKG2D ligand soluble MIC (sMIC) reprograms NK cell to secrete pro-tumorigenic cytokines with diminished cytotoxicity and polyfunctional potential. Antibody clearing sMIC restores NK cell to a normal cytotoxic effector functional state. We discovered that sMIC selectively activates the CBM-signalosome inflammatory pathways in NK cells. Conversely, tumor cell membrane-bound MIC (mMIC) stimulates NK cell cytotoxicity through activating PLC2γ2/SLP-76/Vav1 pathway. Ultimately, antibody targeting sMIC effectuated the in vivo anti-tumor effect of adoptively transferred NK cells. Our findings uncover an unrecognized mechanism that could instruct NK cell to a dysfunctional state in response to cues in the tumor microenvironment. Our findings provide a rationale for co-targeting sMIC to enhance the efficacy of the ongoing NK cell-based cancer immunotherapy.

Differential Effects of Histone Deacetylases on the Expression of NKG2D Ligands and NK Cell-Mediated Anticancer Immunity in Lung Cancer Cells

Histone acetylation is an epigenetic mechanism that regulates the expression of various genes, such as natural killer group 2, member D (NKG2D) ligands. These NKG2D ligands are the key molecules that activate immune cells expressing the NKG2D receptor. It has been observed that cancer cells overexpress histone deacetylases (HDACs) and show reduced acetylation of nuclear histones. Furthermore, HDAC inhibitors are known to upregulate the expression of NKG2D ligands. Humans have 18 known HDAC enzymes that are divided into four classes. At present, it is not clear which types of HDAC are involved in the expression of NKG2D ligands. We hypothesized that specific types of HDAC genes might be responsible for altering the expression of NKG2D ligands. In this study, we monitored the expression of NKG2D ligands and major histocompatibility complex (MHC) class I molecules in lung cancer cells which were treated with six selective HDAC inhibitors and specific small interfering RNAs (siRNAs). We observed that treatment with FK228, which is a selective HDAC1/2 inhibitor, also known as Romidepsin, induced NKG2D ligand expression at the transcriptional and proteomic levels in two different lung cancer cell lines. It also caused an increase in the susceptibility of NCI-H23 cells to NK cells. Silencing HDAC1 or HDAC2 using specific siRNAs increased NKG2D ligand expression. In conclusion, it appears that HDAC1 and HDAC2 might be the key molecules regulating the expression of NKG2D ligands. These results imply that specifically inhibiting HDAC1 and HDAC2 could induce the expression of NKG2D ligands and improve the NK cell-mediated anti-cancer immunity.

Memory CD8 T Cells Generated by Cytomegalovirus Vaccine Vector Expressing NKG2D Ligand Have Effector-Like Phenotype and Distinct Functional Features

Viral vectors have emerged as a promising alternative to classical vaccines due to their great potential for induction of a potent cellular and humoral immunity. Cytomegalovirus (CMV) is an attractive vaccine vector due to its large genome with many non-essential immunoregulatory genes that can be easily manipulated to modify the immune response. CMV generates a strong antigen-specific CD8 T cell response with a gradual accumulation of these cells in the process called memory inflation. In our previous work, we have constructed a mouse CMV vector expressing NKG2D ligand RAE-1γ in place of its viral inhibitor m152 (RAE-1γMCMV), which proved to be highly attenuated in vivo. Despite attenuation, RAE-1γMCMV induced a substantially stronger CD8 T cell response to vectored antigen than the control vector and provided superior protection against bacterial and tumor challenge. In the present study, we confirmed the enhanced protective capacity of RAE-1γMCMV as a tumor vaccine vector and determined the phenotypical and functional characteristics of memory CD8 T cells induced by the RAE-1γ expressing MCMV. RNAseq data revealed higher transcription of numerous genes associated with effector-like CD8 T cell phenotype in RAE-1γMCMV immunized mice. CD8 T cells primed with RAE-1γMCMV were enriched in TCF1 negative population, with higher expression of KLRG1 and lower expression of CD127, CD27, and Eomes. These phenotypical differences were associated with distinct functional features as cells primed with RAE-1γMCMV showed inferior cytokine-producing abilities but comparable cytotoxic potential. After adoptive transfer into naive hosts, OT-1 cells induced with both RAE-1γMCMV and the control vector were equally efficient in rejecting established tumors, suggesting the context of latent infection and cell numbers as important determinants of enhanced anti-tumor response following RAE-1γMCMV vaccination. Overall, our results shed new light on the phenotypical and functional distinctness of memory CD8 T cells induced with CMV vector expressing cellular ligand for the NKG2D receptor.

Pseudorabies Virus Infection Causes Downregulation of Ligands for the Activating NK Cell Receptor NKG2D

Herpesviruses display a complex and carefully balanced interaction with important players in the antiviral immune response of immunocompetent natural hosts, including natural killer (NK) cells. With regard to NK cells, this delicate balance is illustrated on the one hand by severe herpesvirus disease reported in individuals with NK cell deficiencies and on the other hand by several NK cell evasion strategies described for herpesviruses. In the current study, we report that porcine cells infected with the porcine alphaherpesvirus pseudorabies virus (PRV) display a rapid and progressive downregulation of ligands for the major activating NK cell receptor NKG2D. This downregulation consists both of a downregulation of NKG2D ligands that are already expressed on the cell surface of an infected cell and an inhibition of cell surface expression of newly expressed NKG2D ligands. Flow cytometry and RT-qPCR assays showed that PRV infection results in downregulation of the porcine NKG2D ligand pULBP1 from the cell surface and a very substantial suppression of mRNA expression of pULBP1 and of another potential NKG2D ligand, pMIC2. Furthermore, PRV-induced NKG2D ligand downregulation was found to be independent of late viral gene expression. In conclusion, we report that PRV infection of host cells results in a very pronounced downregulation of ligands for the activating NK cell receptor NKG2D, representing an additional NK evasion strategy of PRV.

Chimeric Antigen Receptor T Cells Targeting NKG2D-Ligands Show Robust Efficacy Against Acute Myeloid Leukemia and T-Cell Acute Lymphoblastic Leukemia

CAR T cell approaches to effectively target AML and T-ALL without off-tumor effects on healthy myeloid or T cell compartments respectively are an unmet medical need. NKG2D-ligands are a promising target given their absence on healthy cells and surface expression in a wide range of malignancies. NKG2D-ligand expression has been reported in a substantial group of patients with AML along with evidence for prognostic significance. However, reports regarding the prevalence and density of NKG2D-ligand expression in AML vary and detailed studies to define whether low level expression is sufficient to trigger NKG2D-ligand directed CART cell responses are lacking. NKG2D ligand expression in T-ALL has not previously been interrogated. Here we report that NKG2D-ligands are expressed in T-ALL cell lines and primary T-ALL. We confirm that NKG2D-ligands are frequently surface expressed in primary AML, albeit at relatively low levels. Utilizing CAR T cells incorporating the natural immune receptor NKG2D as the antigen binding domain, we demonstrate striking in vitro activity of CAR T cells targeting NKG2D-ligands against AML and T-ALL cell lines and show that even low-level ligand expression in primary AML targets results in robust NKG2D-CAR activity. We found that NKG2D-ligand expression can be selectively enhanced in low-expressing AML cell lines and primary AML blasts via pharmacologic HDAC inhibition. Such pharmacologic NKG2D-ligand induction results in enhanced NKG2D-CAR anti-leukemic activity without affecting healthy PBMC, thereby providing rationale for the combination of HDAC-inhibitors with NKG2D-CAR T cell therapy as a potential strategy to achieve clinical NKG2D-CAR T cell efficacy in AML.