Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4+ T-cells for the treatment of Hyper IgM1.

Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4+ T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo.

Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand (CD40LG) gene with a median survival of 25 years, potentially treatable with in situ CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of CD40LG in edited healthy donor and patient cells recapitulating the physiological CD40LG regulation. We then documented the preserved fitness of edited cells by xenotransplantation into immunodeficient mice. Finally, we transitioned to large-scale manufacturing, and developed a panel of quality control assays. Overall, our GMP-compliant process takes long-range gene editing one step closer to clinical application with a reassuring safety profile.

Characterization and Functional Analysis of CD44v6.CAR T Cells Endowed with a New Low-Affinity Nerve Growth Factor Receptor-Based Spacer.

Effectiveness of adoptively transferred chimeric antigen receptor (CAR) T cells strongly depends on the quality of CAR-mediated interaction of the effector cells with the target antigen on tumor cells. A major role in this interaction is played by the affinity of the single-chain variable fragment (scFv) for the antigen, and by the CAR design. In particular, the spacer domain may impact on the CAR T cell function by affecting the length and flexibility of the resulting CAR. This study addresses the need to improve the manufacturing process and the antitumor activity of CD44v6-specific CAR T cells by defining the optimal structure of a spacer region derived from the extracellular domain of the human low-affinity nerve growth factor receptor (LNGFR). We tailored the LNGFR spacer to modulate CAR length to efficiently recognize distal or proximal epitopes and to allow selection of transduced CAR T cells by the use of clinical-grade validated manufacturing systems. The different LNGFR spacers investigated in this study are responsible for the generation of CAR T cells with a different memory phenotype, which is mainly related to the level of CAR expression and the extent of the associated tonic signaling. In particular, the CD44v6-NWN2.CAR T cells are enriched in central memory cells and show improved in vitro functions in terms of killing capability, and in vivo antitumor activity against hematological and solid tumors. Clinical Trial Registration numbers: clinicaltrial.gov NCT04097301; ClinicalTrials.gov, NCT00423124.

CAR T Cells Redirected to CD44v6 Control Tumor Growth in Lung and Ovary Adenocarcinoma Bearing Mice.

The main challenge of adoptive therapy with Chimeric Antigen Receptor modified T cells (CAR T) is the application to the field of solid tumors, where the identification of a proper antigen has emerged as one of the major drawbacks to CAR T cell treatment success. CD44 is a glycoprotein involved in cell-cell and cell-matrix interactions. The isoform containing the variant domain 6 of CD44 gene (CD44v6) has been implicated in tumorigenesis, tumor cell invasion and metastasis and represents an attractive target for CAR T cell therapies. Targeting CD44v6 antigen has been shown to control tumor growth in acute myeloid leukemia and multiple myeloma mouse models. While CAR T approach for the treatment of B cell malignancies has shown great success, response rates among patients with solid cancer are less favorable. The purpose of our study was to test the efficacy of CD44v6.CAR T cells, produced in compliance with Good Manufacturing Practice (GMP), in adenocarcinoma tumor models. We generated a bicistronic retroviral vector containing the CD44v6 CAR and the HSV-TK Mut2 suicide gene to enhance the safety of the proposed CAR T cell therapy. CD44v6 transduced CAR T cells were homogeneously positive for ΔLNGFR selection marker, were enriched in T central memory (TCM) and T memory stem cells (TSCM) and displayed a highly activated phenotype. In vitro assays revealed antigen-specific activation and cytotoxicity of human CD44v6.CAR T cells against CD44v6 expressing tumor cell lines. When infused in immunodeficient tumor bearing mice, human CD44v6.CAR T cells were able to reach, infiltrate and proliferate at tumor sites, finally resulting in tumor growth control. Next, we checked if cells produced in compliance with GMP grade standards retained the same antitumor activity of those produced with research grade materials and protocols. Noteworthy, no differences in the potency of the CAR T obtained with the two manufacturing processes were observed. In conclusion, our preclinical results suggest that CD44v6.CAR T based adoptive therapy could be a promising strategy in solid cancer treatment.

Mechanism of Action of the Tumor Vessel Targeting Agent NGR-hTNF: Role of Both NGR Peptide and hTNF in Cell Binding and Signaling.

NGR-hTNF is a therapeutic agent for a solid tumor that specifically targets angiogenic tumor blood vessels, through the NGR motif. Its activity has been assessed in several clinical studies encompassing tumors of different histological types. The drug's activity is based on an improved permeabilization of newly formed tumor vasculature, which favors intratumor penetration of chemotherapeutic agents and leukocyte trafficking. This work investigated the binding and the signaling properties of the NGR-hTNF, to elucidate its mechanism of action. The crystal structure of NGR-hTNF and modeling of its interaction with TNFR suggested that the NGR region is available for binding to a specific receptor. Using 2D TR-NOESY experiments, this study confirmed that the NGR-peptides binds to a specific CD13 isoform, whose expression is restricted to tumor vasculature cells, and to some tumor cell lines. The interaction between hTNF or NGR-hTNF with immobilized TNFRs showed similar kinetic parameters, whereas the competition experiments performed on the cells expressing both TNFR and CD13 showed that NGR-hTNF had a higher binding affinity than hTNF. The analysis of the NGR-hTNF-triggered signal transduction events showed a specific impairment in the activation of pro-survival pathways (Ras, Erk and Akt), compared to hTNF. Since a signaling pattern identical to NGR-hTNF was obtained with hTNF and NGR-sequence given as distinct molecules, the inhibition observed on the survival pathways was presumably due to a direct effect of the NGR-CD13 engagement on the TNFR signaling pathway. The reduced activation of the pro survival pathways induced by NGR-hTNF correlated with the increased caspases activation and reduced cell survival. This study demonstrates that the binding of the NGR-motif to CD13 determines not only the homing of NGR-hTNF to tumor vessels, but also the increase in its antiangiogenic activity.

Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene.

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

The tumor vessel targeting agent NGR-TNF controls the different stages of the tumorigenic process in transgenic mice by distinct mechanisms.

NGR-TNF is a vascular targeting agent in advanced clinical development, coupling tumor necrosis factor-α (TNF) with the CNGRCG peptide, which targets a CD13 isoform specifically expressed by angiogenic vessels. Antitumor efficacy of NGR-TNF has been described in different transplantation tumor models. Nevertheless, the mechanism underlying its activity is not fully understood. In the wild type and in the immunodeficient (RAG-/-) RIP1-Tag2 models of multistage pancreatic carcinogenesis, we demonstrate that CD13 is highly expressed on endothelial cells of hyperplastic and angiogenic islets, whereas its expression is down regulated in tumors where it partially colocalize with pericytes. In vivo CNGRCG peptides coupled to fluorescent nanoparticles (quantum dots) bind to CD13 and colocalize with anti-CD31, in pancreatic islets. At early stage, low doses of NGR-murine (m)TNF have a direct cytotoxic effect inducing endothelial cell apoptosis, reducing vessel density and eventually inhibiting the development of angiogenic islets. At a later stage, NGR-mTNF is able to reduce tumor growth inducing vascular normalization, exclusively when treatment is carried out in the immunocompetent mice. Interestingly, NGR-mTNF-treated tumors from these mice are characterized by CD8+ T cell infiltration. At molecular level, overexpression of genes involved in vessels normalization was detected only in NGR-mTNF-treated tumors from immunocompetent mice. These findings identified a new mechanism of action of NGR-mTNF, providing support for the development of new therapeutic strategies combining chemotherapy or active/adoptive immunotherapies to low dose NGR-TNF treatment.

Anti-metastatic activity of the tumor vascular targeting agent NGR-TNF.

Tumor vessels are an attractive target for cancer therapy, including metastasis treatment. Angiogenesis inhibitors targeting the VEGF signalling pathway have proven to be efficacious in preclinical cancer models and in clinical trials. However, angiogenesis inhibition concomitantly elicits tumor adaptation and progression to stages of greater malignancy, with heightened invasiveness and in some cases increased distant metastasis. Here, we investigated whether NGR-TNF, a vascular targeting agent in phase III clinical development, coupling the CNGRCG angiogenic vessel-homing peptide with TNF-α, has an effect on metastasis in a model of murine breast cancer, which spontaneously metastasize to lungs, and on the growth of experimental melanoma lung metastasis. We report that NGR-TNF does not increase cancer invasiveness, as other antiangiogenics agents do, but controls metastatic growth in both models, both when administered as primary treatment and in adjuvant settings, improving the overall survival of metastasis-bearing mice.

Therapeutic genes for anti-HIV/AIDS gene therapy.

The multiple therapeutic approaches developed so far to cope HIV-1 infection, such as anti-retroviral drugs, germicides and several attempts of therapeutic vaccination have provided significant amelioration in terms of life-quality and survival rate of AIDS patients. Nevertheless, no approach has demonstrated efficacy in eradicating this lethal, if untreated, infection. The curative power of gene therapy has been proven for the treatment of monogenic immunodeficiensies, where permanent gene modification of host cells is sufficient to correct the defect for life-time. No doubt, a similar concept is not applicable for gene therapy of infectious immunodeficiensies as AIDS, where there is not a single gene to be corrected; rather engineered cells must gain immunotherapeutic or antiviral features to grant either short- or long-term efficacy mostly by acquisition of antiviral genes or payloads. Anti-HIV/AIDS gene therapy is one of the most promising strategy, although challenging, to eradicate HIV-1 infection. In fact, genetic modification of hematopoietic stem cells with one or multiple therapeutic genes is expected to originate blood cell progenies resistant to viral infection and thereby able to prevail on infected unprotected cells. Ultimately, protected cells will re-establish a functional immune system able to control HIV-1 replication. More than hundred gene therapy clinical trials against AIDS employing different viral vectors and transgenes have been approved or are currently ongoing worldwide. This review will overview anti-HIV-1 infection gene therapy field evaluating strength and weakness of the transgenes and payloads used in the past and of those potentially exploitable in the future.

Selective preservation of bone marrow mature recirculating but not marginal zone B cells in murine models of chronic inflammation.

Inflammation promotes granulopoiesis over B lymphopoiesis in the bone marrow (BM). We studied B cell homeostasis in two murine models of T cell mediated chronic inflammation, namely calreticulin-deficient fetal liver chimeras (FLC), which develop severe blepharitis and alopecia due to T cell hyper responsiveness, and inflammatory bowel disease (IBD) caused by injection of CD4(+) naïve T cells into lymphopenic mice. We show herein that despite the severe depletion of B cell progenitors during chronic, peripheral T cell-mediated inflammation, the population of BM mature recirculating B cells is unaffected. These B cells are poised to differentiate to plasma cells in response to blood borne pathogens, in an analogous fashion to non-recirculating marginal zone (MZ) B cells in the spleen. MZ B cells nevertheless differentiate more efficiently to plasma cells upon polyclonal stimulation by Toll-like receptor (TLR) ligands, and are depleted during chronic T cell mediated inflammation in vivo. The preservation of mature B cells in the BM is associated with increased concentration of macrophage migration inhibitory factor (MIF) in serum and BM plasma. MIF produced by perivascular dendritic cells (DC) in the BM provides a crucial survival signal for recirculating B cells, and mice treated with a MIF inhibitor during inflammation showed significantly reduced mature B cells in the BM. These data indicate that MIF secretion by perivascular DC may promote the survival of the recirculating B cell pool to ensure responsiveness to blood borne microbes despite loss of the MZ B cell pool that accompanies depressed lymphopoiesis during inflammation.

Chim3 confers survival advantage to CD4+ T cells upon HIV-1 infection by preventing HIV-1 DNA integration and HIV-1-induced G2 cell-cycle delay.

The long-term expression and the ability of a therapeutic gene to confer survival advantage to transduced cells are mandatory requirements for successful anti-HIV gene therapy. In this context, we developed lentiviral vectors (LVs) expressing the F12-viral infectivity factor (Vif) derivative Chim3. We recently showed that Chim3 inhibits HIV-1 replication in primary cells by both blocking the accumulation of retrotranscripts, independently of either human APOBEC3G (hA3G) or Vif, and by preserving the antiviral function of hA3G. These results were predictive of long-lasting survival of Chim3(+) cells after HIV-1 infection. Furthermore, Vif, like Vpr, deregulates cell-cycle progression by inducing a delay in G(2) phase. Thus, the aim of this study was to investigate the role of Chim3 on both cell survival and cell-cycle regulation after HIV-1 infection. Here, we provide evidence that infected Chim3(+) T cells prevail over either mock- or empty-LV engineered cells, show reduced G(2) accumulation, and, as a consequence, ultimately extend their lifespan. Based on these findings, Chim3 rightly belongs to the most efficacious class of antiviral genes. In conclusion, Chim3 usage in anti-HIV gene therapy based on hematopoietic stem cell (HSC) modification has to be considered as a promising therapeutic intervention to eventually cope with HIV-1 infection.

Mutational analysis of the HIV-1 auxiliary protein Vif identifies independent domains important for the physical and functional interaction with HIV-1 reverse transcriptase.

The HIV-1 accessory protein Vif plays a dual role: it counteracts the natural restriction factors APOBEC3G and 3F and ensures efficient retrotranscription of the HIV-1 RNA genome. We have previously shown that Vif can act as an auxiliary factor for HIV-1 reverse transcriptase (RT), increasing its rate of association to RNA or DNA templates. Here, by using seven different Vif mutants, we provide in vitro evidences that Vif stimulates HIV-1 RT through direct protein-protein interaction, which is mediated by its C-terminal domain. Physical interaction appears to require the proline-rich region comprised between amino acid (aa) 161 and 164 of Vif, whereas the RT stimulatory activity requires, in addition, the extreme C-terminal region (aa 169-192) of the Vif protein. Neither the RNA interaction domain, nor the Zn(++)-binding domain of Vif are required for its interaction with the viral RT. Pseudotyped HIV-1 lentiviral vectors bearing Vif mutants deleted in the RNA- or RT-binding domains show defects in retrotranscription/integration processes in both permissive and nonpermissive cells. Our results broaden our knowledge on how three important functions of Vif (RNA binding, RT binding and stimulation and Zn(++) binding), are coordinated by different domains.

Transcriptional enhancers induce insertional gene deregulation independently from the vector type and design.

The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) gamma-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase--PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a gamma-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design.

The F12-Vif derivative Chim3 inhibits HIV-1 replication in CD4+ T lymphocytes and CD34+-derived macrophages by blocking HIV-1 DNA integration.

The viral infectivity factor (Vif) is essential for HIV-1 infectivity and hence is an ideal target for promising anti-HIV-1/AIDS gene therapy. We previously demonstrated that F12-Vif mutant inhibits HIV-1 replication in CD4(+) T lymphocytes. Despite macrophage relevance to HIV-1 pathogenesis, most gene therapy studies do not investigate macrophages because of their natural resistance to genetic manipulation. Here, we confirm the F12-Vif antiviral activity also in macrophages differentiated in vitro from transduced CD34(+) human stem cells (HSCs). Moreover, we identified the 126- to 170-amino-acid region in the C-terminal half of F12-Vif as responsible for its antiviral function. Indeed, Chim3 protein, containing this 45-amino-acid region embedded in a WT-Vif backbone, is as lethal as F12-Vif against HIV-1. Of major relevance, we demonstrated a dual mechanism of action for Chim3. First, Chim3 functions as a transdominant factor that preserves the antiviral function of the natural restriction factor APOBEC3G (hA3G). Second, Chim3 blocks the early HIV-1 retrotranscript accumulation and thereby HIV-1 DNA integration regardless of the presence of WT-Vif and hA3G. In conclusion, by impairing the early steps of HIV-1 life cycle, Chim3 conceivably endows engineered cells with survival advantage, which is required for the efficient immune reconstitution of patients living with HIV/AIDS.

Recombinase-deficient T cell development by selective accumulation of CD3 into lipid rafts.

The pre-T cell receptor (pre-TCR) promotes the development of thymocytes with productive rearrangement at the TCR beta chain locus by signaling in a ligand-independent fashion. The TCR beta chain associates with the invariant pre-Talpha (pTalpha) chain, which bears specific charged residues in the extracellular portion mediating pre-TCR self-oligomerization. In recombinase-deficient thymocytes, calnexin (CNX) associated with CD3 chains is inefficiently retained in the endoplasmic reticulum (ER) and weakly expressed in the plasma membrane. Deliberate cross-linking of CNX/CD3 complexes mimics pre-TCR signaling. Here, we show that, analogously to the pTalpha chain, surface CNX is palmitoylated and that CD3 prominently accumulated in lipid rafts upon cross-linking. Mutant CNX isoforms devoid of ER retention determined pre-TCR-like signaling and simulated beta selection only when stably translocating CD3 to lipid rafts. Inclusion of the palmitoylated cytoplasmic tail from the pTalpha chain in recombinant CNX strikingly improved the pre-TCR-like signaling efficiency of CNX/CD3 in rafts. This study indicates that lipid rafts in the plasma membrane represent proficient microdomains for the initiation of pre-TCR signaling, and supports the view that beta selection by oligomerized pre-TCR is implemented by the pTalpha cytoplasmic tail.

EVA regulates thymic stromal organisation and early thymocyte development.

Epithelial V-like antigen (EVA) is an immunoglobulin-like adhesion molecule identified in a screen for molecules developmentally regulated at the DN to DP progression in thymocyte development. We show that EVA is expressed during the early stages of thymus organogenesis in both fetal thymic epithelia and T cell precursors, and is progressively downregulated from day 16.5 of embryonic development. In the postnatal thymus, EVA expression is restricted to epithelial cells and is distributed throughout both cortical and medullary thymic regions. Transgenic overexpression of EVA in the thymus cortex resulted in a modified stromal environment, which elicited an increase in organ size and absolute cell number. Although peripheral T lymphocyte numbers are augmented throughout life, no imbalance either in the repertoire, or in the different T cell subsets was detected. Collectively, these data suggest a role for EVA in structural organisation of the thymus and early lymphocyte development.

Regulation of peripheral T cell activation by calreticulin.

Regulated expression of positive and negative regulatory factors controls the extent and duration of T cell adaptive immune response preserving the organism's integrity. Calreticulin (CRT) is a major Ca2+ buffering chaperone in the lumen of the endoplasmic reticulum. Here we investigated the impact of CRT deficiency on T cell function in immunodeficient mice reconstituted with fetal liver crt-/- hemopoietic progenitors. These chimeric mice displayed severe immunopathological traits, which correlated with a lower threshold of T cell receptor (TCR) activation and exaggerated peripheral T cell response to antigen with enhanced secretion of inflammatory cytokines. In crt-/- T cells TCR stimulation induced pulsatile cytosolic elevations of Ca2+ concentration and protracted accumulation of nuclear factor of activated T cells in the nucleus as well as sustained activation of the mitogen-activated protein kinase pathways. These observations support the hypothesis that CRT-dependent shaping of Ca2+ signaling critically contributes to the modulation of the T cell adaptive immune response.

NKG2D-independent suppression of T cell proliferation by H60 and MICA.

The activating receptor NKG2D recognizes a wide range of different ligands, some of which are primarily expressed in "stressed" tissues or on tumor cells. Until now, similar stimulatory effects on natural killer and CD8+ T cells have been described for all NKG2D ligands, and the NKG2D receptor/ligand system has therefore been interpreted as a sensor system involved in tumor immune surveillance and activation of immune responses. We show here that the NKG2D ligands H60 and MIC class 1 chain-related protein A (MICA) can also mediate strong suppressive effects on T cell proliferation. Responsiveness to H60- and MICA-mediated suppression requires IL-10 and involves a receptor other than NKG2D. These findings might provide explanations for the observation that strong in vivo NKG2D ligand expression, such as that on tumor cells, sometimes fails to support effective immune responses and links this observation to a distinct subgroup of NKG2D ligands.