Helmut R. Salih

Fc-Optimized NKG2D-Fc Constructs Induce NK Cell Antibody-Dependent Cellular Cytotoxicity against Breast Cancer Cells Independently of HER2/neu Expression Status

The ability of NK cells to mediate Ab-dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of antitumor Abs, including trastuzumab, which is approved for the treatment of breast cancer with HER2/neu overexpression. Notably, only ∼25% of breast cancer patients overexpress HER2/neu. Moreover, HER2/neu is expressed on healthy cells, and trastuzumab application is associated with side effects. In contrast, the ligands of the activating immunoreceptor NKG2D (NKG2DL) are selectively expressed on malignant cells. In this study, we took advantage of the tumor-associated expression of NKG2DL by using them as target Ags for NKG2D-IgG1 fusion proteins optimized by amino acid exchange S239D/I332E in their Fc part. Compared to constructs with wild-type Fc parts, fusion proteins carrying the S239D/I332E modification (NKG2D–Fc–ADCC) mediated highly enhanced degranulation, ADCC, and IFN-γ production of NK cells in response to breast cancer cells. NKG2D–Fc–ADCC substantially enhanced NK reactivity also against HER2/neu-low targets that were unaffected by trastuzumab, as both compounds mediated their immunostimulatory effects in strict dependence of target Ag expression levels. Thus, in line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16, NKG2D–Fc–ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D–Fc–ADCC may constitute an attractive means for immunotherapy especially of HER2/neu-low or -negative breast cancer.

Long-term observation reveals high-frequency engraftment of human acute myeloid leukemia in immunodeficient mice

Repopulation of immunodeficient mice remains the primary method for functional assessment of human acute myeloid leukemia. Published data report engraftment in ~40–66% of cases, mostly of intermediate- or poor-risk subtypes. Here we report that extending follow-up beyond the standard analysis endpoints of 10 to 16 weeks after transplantation permitted leukemic engraftment from nearly every case of xenotransplanted acute myeloid leukemia (18/19, ~95%). Xenogeneic leukemic cells showed conserved immune pheno-types and genetic signatures when compared to corresponding pre-transplant cells and, furthermore, were able to induce leukemia in re-transplantation assays. Importantly, bone marrow biopsies taken at standardized time points failed to detect leukemic cells in 11/18 of cases that later showed robust engraftment (61%, termed “long-latency engrafters”), indicating that leukemic cells can persist over months at undetectable levels without losing disease-initiating properties. Cells from favorable-risk leukemia subtypes required longer to become detectable in NOD/SCID/IL2Rγnull mice (27.5±9.4 weeks) than did cells from intermediate-risk (21.9±9.4 weeks, P<0.01) or adverse-risk (17±7.6 weeks; P<0.0001) subtypes, explaining why the engraftment of the first was missed with previous protocols. Mechanistically, leukemic cells engrafting after a prolonged latency showed inferior homing to the bone marrow. Finally, we applied our model to favorable-risk acute myeloid leukemia with inv(16); here, we showed that CD34+ (but not CD34−) blasts induced robust, long-latency engraftment and expressed enhanced levels of stem cell genes. In conclusion, we provide a model that allows in vivo mouse studies with a wide range of molecular subtypes of acute myeloid leukemia subtypes which were previously considered not able to engraft, thus enabling novel insights into leukemogenesis.

Graft versus self (GvS) against T-cell autoantigens is a mechanism of graft–host interaction

Significance As the mechanism of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), recognition of the recipient’s body by donor immune cells was previously believed to be based on genetic and immunological differences between donor and recipient. However, evidence in murine models and in autologous HCT has shown that also autoimmunity contributes to GVHD. In this study, we show the development of auto-reactivity after human allogeneic HCT and characterize the specific self-epitopes of T cells that may contribute to mediation of GVHD. Such autoantigens have never been characterized before. These observations contribute to a better understanding of the immune responses that are activated following hematopoietic cell transplantation. Graft-versus-host disease (GVHD) represents the major nonrelapse complication of allogeneic hematopoietic cell transplantation. Although rare, the CNS and the eye can be affected. In this study, manifestation in the retina as part of the CNS and T-cell epitopes recognized by the allogeneic T cells were evaluated. In 2 of 6 patients with posttransplantation retina diseases and 6 of 22 patients without ocular symptoms, antigen-specific T-cell responses against retina-specific epitopes were observed. No genetic differences between donor and recipient could be identified indicating T-cell activation against self-antigens (graft versus self). Transplantation of a preexisting immunity and cross-reactivity with ubiquitous epitopes was excluded in family donors and healthy individuals. In summary, an immunological reaction against retina cells represents a mechanism of graft-versus-host interaction following hematopoietic cell transplantation.

Platelet-mediated shedding of NKG2D ligands impairs NK cell immune-surveillance of tumor cells

ABSTRACT Platelets promote metastasis, among others by coating cancer cells traveling through the blood, which results in protection from NK cell immune-surveillance. The underlying mechanisms, however, remain to be fully elucidated. Here we report that platelet-coating reduces surface expression of NKG2D ligands, in particular MICA and MICB, on tumor cells, which was mirrored by enhanced release of their soluble ectodomains. Similar results were obtained upon exposure of tumor cells to platelet-releasate and can be attributed to the sheddases ADAM10 and ADAM17 that are detectable on the platelet surface and in releasate following activation and at higher levels on platelets of patients with metastasized lung cancer compared with healthy controls. Platelet-mediated NKG2DL-shedding in turn resulted in impaired “induced self” recognition by NK cells as revealed by diminished NKG2D-dependent lysis of tumor cells. Our results indicate that platelet-mediated NKG2DL-shedding may be involved in immune-evasion of (metastasizing) tumor cells from NK cell reactivity.

The Immune Checkpoint Modulator OX40 and Its Ligand OX40L in NK-Cell Immunosurveillance and Acute Myeloid Leukemia

The TNFR-family member OX40 is expressed on AML cells, influencing various AML cellular functions as well as immunosurveillance by OX40L-expressing NK cells. These effects should be considered when developing OX40-targeted approaches for cancer immunotherapy. The TNF receptor family member OX40 promotes activation and proliferation of T cells, which fuels efforts to modulate this immune checkpoint to reinforce antitumor immunity. Besides T cells, NK cells are a second cytotoxic lymphocyte subset that contributes to antitumor immunity, particularly in leukemia. Accordingly, these cells are being clinically evaluated for cancer treatment through multiple approaches, such as adoptive transfer of ex vivo expanded polyclonal NK cells (pNKC). Here, we analyzed whether and how OX40 and its ligand (OX40L) influence NK-cell function and antileukemia reactivity. We report that OX40 is expressed on leukemic blasts in a substantial percentage of patients with acute myeloid leukemia (AML) and that OX40 can, after stimulation with agonistic OX40 antibodies, mediate proliferation and release of cytokines that act as growth and survival factors for the leukemic cells. We also demonstrate that pNKC differentially express OX40L, depending on the protocol used for their generation. OX40L signaling promoted NK-cell activation, cytokine production, and cytotoxicity, and disruption of OX40–OX40L interaction impaired pNKC reactivity against primary AML cells. Together, our data implicate OX40/OX40L in disease pathophysiology of AML and in NK-cell immunosurveillance. Our findings indicate that effects of the OX40–OX40L receptor–ligand system in other immune cell subsets and also malignant cells should be taken into account when developing OX40-targeted approaches for cancer immunotherapy. Cancer Immunol Res; 6(2); 209–21. ©2018 AACR.

The novel deubiquitinase inhibitor b-AP15 induces direct and NK cell-mediated antitumor effects in human mantle cell lymphoma

The first therapeutic proteasome inhibitor bortezomib has clinical efficacy in mantle cell lymphoma (MCL) which resulted in its incorporation in treatment algorithms for this disease. Impairment of proteasomal function by bortezomib is mediated via inhibition of the 20S core particle. However, proteasome function can also be modified by targeting upstream components of the ubiquitin–proteasome system. Recently, b-AP15 has been identified as a small molecule achieving proteasome inhibition by targeting the deubiquitinase (DUB) activity of the 19S regulatory subunit and was found to inhibit cancer cell growth in preclinical analyses. In the present study, both direct antitumor effects and the possibility to induce natural killer group 2 member D ligands (NKG2DL) to reinforce NK cell immunity with b-AP15 were investigated to provide a rational basis for clinical evaluation of this novel DUB inhibitor in MCL. Treatment with b-AP15 resulted in reduced viability as well as induction of apoptosis in a time- and dose-dependent manner, which could be attributed to caspase activation in MCL cells. In addition, treatment with b-AP15 differentially induced NKG2DL expression and subsequent NK cell lysis of MCL cells. These results indicate that the DUB inhibitor b-AP15 displays substantial antitumor activity in human MCL and suggest that b-AP15 might be a novel therapeutic option in the treatment of MCL that warrants clinical investigation.

Expression of 4-1BB and its ligand on blasts correlates with prognosis of patients with AML

Costimulatory ligands (COLs) and their receptors (COR) regulate immune reactions and cellular survival and might be relevant in acute myeloid leukemia (AML). This study evaluated the clinical relevance of 4-1BBL, glucocorticoid-induced TNFR-related protein (GITR) and ligand (GITRL), CD80, and CD86 in case of expression on AML blasts. 98 patients were evaluated at initial diagnosis. Immunophenotypically evaluated specific fluorescence index (SFI) levels of COR and COL on blasts were correlated with morphological, cytogenetic, and several prognostic parameters. Significantly higher COR expression was seen in monocytic versus non-monocytic AML subtypes; GITR, p=0.05; GITRL, p=0.005; CD86, p=0.001). Cut-off values for two COR and their ligands were evaluated: cases presenting with 4-1BB values above cut-off 1.2 SFI levels correlated (tendentially) significantly with a higher probability for disease-free survival (DFS, p=0.06) and a favorable HR of 0.2; p=0.04 for relapse. HR for death was also significantly lower in this group (0.12; p=0.04). In contrast, a lower probability for DFS and overall survival was seen in cases with 4-1BBL expression above 2.2 SFI levels (p=0.08 and p=0.09). In addition, multivariate analysis showed a significantly higher probability of death in this group (HR 10.3, p=0.04). Expression of CD80 and CD86 did not show significant prognostic relevance. On initial diagnosis, 4-1BB and 4-1BBL qualify as markers for prediction of patients’ course and represent a valuable screening target for patients with AML at initial diagnosis.

The BCR-ABL inhibitor nilotinib influences phenotype and function of monocyte-derived human dendritic cells

In chronic myeloid leukemia (CML), the translocation t(9;22) results in the fusion protein BCR-ABL (breakpoint cluster region-abelson murine leukemia), a tyrosine kinase mediating oncogenic signaling which is successfully targeted by treatment with BCR-ABL inhibitors like imatinib. However, BCR-ABL inhibitors may also affect antitumor immunity. For instance, it was reported that imatinib impairs the function of dendritic cells (DCs) that play a central role in initiating and sustaining T cell responses. Meanwhile, second generation BCR-ABL inhibitors like nilotinib, which inhibits BCR-ABL with enhanced potency have become standard of treatment, at least in patients with BCR-ABL kinase domain mutations. In this study we analyzed the influence of therapeutic concentrations of nilotinib on human monocyte-derived DCs and compared its effects to imatinib. We found that both tyrosine kinase inhibitors (TKI) comparably and significantly impaired differentiation of monocytes to DCs as revealed by curtated downregulation of CD14 and reduced upregulation of CD1a and CD83. This was only partially restored after withdrawal of the TKI. Moreover, both TKI significantly reduced activation-induced IL-12p70 and C-C motif chemokine ligand (CCL) 3 secretion, while divergent TKI effects for CCL2 and CCL5 were observed. In contrast, only nilotinib significantly impaired the migratory capacity of DCs and their capacity to induce T-cell immune responses in MLRs. Our results indicate that imatinib and nilotinib may differ significantly with regard to their influence on antitumor immunity. Thus, for future combinatory approaches and particularly stop studies in CML treatment, choice of the most suitable BCR-ABL inhibitor requires careful consideration.