Decitabine enhances targeting of AML cells by CD34+ progenitor-derived NK cells in NOD/SCID/IL2Rgnull mice.

Combining natural killer (NK) cell adoptive transfer with hypomethylating agents (HMAs) is an attractive therapeutic approach for patients with acute myeloid leukemia (AML). However, data regarding the impact of HMAs on NK cell functionality are mostly derived from in vitro studies with high nonclinical relevant drug concentrations. In the present study, we report a comparative study of azacitidine (AZA) and decitabine (DAC) in combination with allogeneic NK cells generated from CD34+ hematopoietic stem and progenitor cells (HSPC-NK cells) in in vitro and in vivo AML models. In vitro, low-dose HMAs did not impair viability of HSPC-NK cells. Furthermore, low-dose DAC preserved HSPC-NK killing, proliferation, and interferon gamma production capacity, whereas AZA diminished their proliferation and reactivity. Importantly, we showed HMAs and HSPC-NK cells could potently work together to target AML cell lines and patient AML blasts. In vivo, both agents exerted a significant delay in AML progression in NOD/SCID/IL2Rgnull mice, but the persistence of adoptively transferred HSPC-NK cells was not affected. Infused NK cells showed sustained expression of most activating receptors, upregulated NKp44 expression, and remarkable killer cell immunoglobulin-like receptor acquisition. Most importantly, only DAC potentiated HSPC-NK cell anti-leukemic activity in vivo. Besides upregulation of NKG2D- and DNAM-1-activating ligands on AML cells, DAC enhanced messenger RNA expression of inflammatory cytokines, perforin, and TRAIL by HSPC-NK cells. In addition, treatment resulted in increased numbers of HSPC-NK cells in the bone marrow compartment, suggesting that DAC could positively modulate NK cell activity, trafficking, and tumor targeting. These data provide a rationale to explore combination therapy of adoptive HSPC-NK cells and DAC in patients with AML.

Increased Coexpression of PD-1, TIGIT, and KLRG-1 on Tumor-Reactive CD8+ T Cells During Relapse after Allogeneic Stem Cell Transplantation.

Allogeneic stem cell transplantation (allo-SCT) can be a curative treatment for patients with a hematologic malignancy due to alloreactive T cell responses recognizing minor histocompatibility antigens (MiHA). Yet tumor immune escape mechanisms can cause failure of T cell immunity, leading to relapse. Tumor cells display low expression of costimulatory molecules and can up-regulate coinhibitory molecules that inhibit T cell functionality on ligation with their counter-receptors on the tumor-reactive T cells. The aim of this explorative study was to evaluate immune checkpoint expression profiles on T cell subsets and on cytomegalovirus (CMV)- and/or MiHA-reactive CD8+ T cells of allo-SCT recipients using a 13-color flow cytometry panel, and to correlate these expression patterns to clinical outcomes. MiHA-reactive CD8+ T cells exhibited an early differentiated CD27++/CD28++ phenotype with low KLRG-1 and CD57 expression. These T cells also displayed increased expression of PD-1, TIM-3, and TIGIT compared with total effector memory T cells and CMV-specific CD8+ T cells in healthy donors and allo-SCT recipients. Remarkably, high coexpression of PD-1, TIGIT, and KLRG-1 on MiHA-reactive CD8+ T cells was associated with relapse after allo-SCT. Taken together, these findings indicate that MiHA-specific CD8+ T cells of relapsed patients have a distinctive coinhibitory expression signature compared with patients who stay in remission. This phenotype may serve as a potential monitoring tool in patients. Moreover, these findings suggest that PD-1 and TIGIT play important roles in regulating T cell-mediated tumor control, providing a rationale for immunotherapy with blocking antibodies to treat relapse after allo-SCT.

Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy.

Effective T-cell therapy against cancer is dependent on the formation of long-lived, stem cell-like T cells with the ability to self-renew and differentiate into potent effector cells. Here, we investigated the in vivo existence of stem cell-like antigen-specific T cells in allogeneic stem cell transplantation (allo-SCT) patients and their ex vivo generation for additive treatment posttransplant. Early after allo-SCT, CD8+ stem cell memory T cells targeting minor histocompatibility antigens (MiHAs) expressed by recipient tumor cells were not detectable, emphasizing the need for improved additive MiHA-specific T-cell therapy. Importantly, MiHA-specific CD8+ T cells with an early CCR7+CD62L+CD45RO+CD27+CD28+CD95+ memory-like phenotype and gene signature could be expanded from naive precursors by inhibiting Akt signaling during ex vivo priming and expansion. This resulted in a MiHA-specific CD8+ T-cell population containing a high proportion of stem cell-like T cells compared with terminal differentiated effector T cells in control cultures. Importantly, these Akt-inhibited MiHA-specific CD8+ T cells showed a superior expansion capacity in vitro and in immunodeficient mice and induced a superior antitumor effect in intrafemural multiple myeloma-bearing mice. These findings provide a rationale for clinical exploitation of ex vivo-generated Akt-inhibited MiHA-specific CD8+ T cells in additive immunotherapy to prevent or treat relapse in allo-SCT patients.

B and T lymphocyte attenuator mediates inhibition of tumor-reactive CD8+ T cells in patients after allogeneic stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) can cure hematological malignancies by inducing alloreactive T cell responses targeting minor histocompatibility antigens (MiHA) expressed on malignant cells. Despite induction of robust MiHA-specific T cell responses and long-term persistence of alloreactive memory T cells specific for the tumor, often these T cells fail to respond efficiently to tumor relapse. Previously, we demonstrated the involvement of the coinhibitory receptor programmed death-1 (PD-1) in suppressing MiHA-specific CD8(+) T cell immunity. In this study, we investigated whether B and T lymphocyte attenuator (BTLA) plays a similar role in functional impairment of MiHA-specific T cells after allo-SCT. In addition to PD-1, we observed higher BTLA expression on MiHA-specific CD8(+) T cells compared with that of the total population of CD8(+) effector-memory T cells. In addition, BTLA's ligand, herpes virus entry mediator (HVEM), was found constitutively expressed by myeloid leukemia, B cell lymphoma, and multiple myeloma cells. Interference with the BTLA-HVEM pathway, using a BTLA blocking Ab, augmented proliferation of BTLA(+)PD-1(+) MiHA-specific CD8(+) T cells by HVEM-expressing dendritic cells. Notably, we demonstrated that blocking of BTLA or PD-1 enhanced ex vivo proliferation of MiHA-specific CD8(+) T cells in respectively 7 and 9 of 11 allo-SCT patients. Notably, in 3 of 11 patients, the effect of BTLA blockade was more prominent than that of PD-1 blockade. Furthermore, these expanded MiHA-specific CD8(+) T cells competently produced effector cytokines and degranulated upon Ag reencounter. Together, these results demonstrate that BTLA-HVEM interactions impair MiHA-specific T cell functionality, providing a rationale for interfering with BTLA signaling in post-stem cell transplantation therapies.

Homing characteristics of donor T cells after experimental allogeneic bone marrow transplantation and posttransplantation therapy for multiple myeloma.

Relapse and graft-versus-host disease remain major problems associated with allogeneic bone marrow (BM) transplantation (allo-BMT) and posttransplantation therapy in patients with multiple myeloma (MM) and other hematologic malignancies. A possible strategy for selectively enhancing the graft-versus-myeloma response and possibly reducing graft-versus-host disease is to increase the migration of alloreactive T cells toward the MM-containing BM. In the present study, we characterized the BM-homing behavior of donor-derived effector T cells in a novel allo-BMT model for the treatment of MM. We observed that posttransplantation immunotherapy consisting of donor lymphocyte infusion (DLI) and vaccination with minor histocompatibility antigen-loaded dendritic cells (DCs) was associated with prolonged survival compared with allo-BMT with no further treatment. Moreover, CD8(+) effector T cells expressing inflammatory homing receptors, including high levels of CD44, LFA-1, and inflammatory chemokine receptors, were recruited to MM-bearing BM. This was paralleled by strongly increased expression of IFN-γ and IFN-γ-inducible chemokines, including CXCL9, CXCL10, and CXCL16, especially in mice treated with DLI plus minor histocompatibility antigen-loaded DC vaccination. Remarkably, expression of the homeostatic chemokine CXCL12 was reduced. Furthermore, IFN-γ and TNF-α induced BM endothelial cells to express high levels of the inflammatory chemokines and reduced or unaltered levels of CXCL12. Finally, presentation of CXCL9 by multiple BM endothelial cell-expressed heparan sulfate proteoglycans triggered transendothelial migration of effector T cells. Taken together, our data demonstrate that both post-transplantation DLI plus miHA-loaded DC vaccination and MM growth result in an increased expression of inflammatory homing receptors on donor T cells, decreased levels of the homeostatic BM-homing chemokine CXCL12, and strong induction of inflammatory chemokines in the BM. Thus, along with increasing the population of alloreactive T cells, post-transplantation immunotherapy also might contribute to a more effective graft-versus-tumor response by switching homeostatic T cell migration to inflammation-driven migration.

Immunogenicity of dendritic cells pulsed with MAGE3, Survivin and B-cell maturation antigen mRNA for vaccination of multiple myeloma patients.

The introduction of autologous stem cell transplantation (SCT) and novel drugs has improved overall survival in multiple myeloma (MM) patients. However, minimal residual disease (MRD) remains and most patients eventually relapse. Myeloma plasma cells express tumor-associated antigens (TAA), which are interesting targets for immunotherapy. In this phase 1 study, we investigated the safety and immunological effects of TAA-mRNA-loaded dendritic cell (DC) vaccination for treatment for MRD in MM after SCT. Mature monocyte-derived DCs were pulsed with keyhole limpet hemocyanin (KLH) and electroporated with MAGE3, Survivin or B-cell maturation antigen (BCMA) mRNA. Twelve patients were vaccinated three times with intravenous (5-22 × 10(6) DCs) and intradermal vaccines (4-11 × 10(6) DCs), at biweekly intervals. Immunological responses were monitored in blood and delayed-type hypersensitivity (DTH) biopsies. All patients developed strong anti-KLH T-cell responses, but not KLH antibodies. In 2 patients, vaccine-specific T cells were detected in DTH biopsies. In one patient, we found MAGE3-specific CD4(+) and CD8(+) T cells, and CD3(+) T cells reactive against BCMA and Survivin. In the other patient, we detected low numbers of MAGE3 and BCMA-reactive CD8(+) T cells. Vaccination was well tolerated with limited toxicity. These findings illustrate that TAA-mRNA-electroporated mature DCs are capable of inducing TAA-T-cell responses in MM patients after SCT.

siRNA silencing of PD-L1 and PD-L2 on dendritic cells augments expansion and function of minor histocompatibility antigen-specific CD8+ T cells.

Tumor relapse after human leukocyte antigen-matched allogeneic stem cell transplantation (SCT) remains a serious problem, despite the long-term presence of minor histocompatibility antigen (MiHA)-specific memory T cells. Dendritic cell (DC)-based vaccination boosting MiHA-specific T-cell immunity is an appealing strategy to prevent or counteract tumor recurrence, but improvement is necessary to increase the clinical benefit. Here, we investigated whether knockdown of programmed death ligand 1 (PD-L1) and PD-L2 on monocyte-derived DCs results in improved T-cell activation. Electroporation of single siRNA sequences into immature DCs resulted in efficient, specific, and long-lasting knockdown of PD-L1 and PD-L2 expression. PD-L knockdown DCs strongly augmented interferon-γ and interleukin-2 production by stimulated T cells in an allogeneic mixed lymphocyte reaction, whereas no effect was observed on T-cell proliferation. Moreover, we demonstrated that PD-L gene silencing, especially combined PD-L1 and PD-L2 knockdown, resulted in improved proliferation and cytokine production of keyhole limpet hemocyanin-specific CD4(+) T cells. Most importantly, PD-L knockdown DCs showed superior potential to expand MiHA-specific CD8(+) effector and memory T cells from leukemia patients early after donor lymphocyte infusion and later during relapse. These data demonstrate that PD-L siRNA electroporated DCs are highly effective in enhancing T-cell proliferation and cytokine production, and are therefore attractive cells for improving the efficacy of DC vaccines in cancer patients.

Myeloid leukemic progenitor cells can be specifically targeted by minor histocompatibility antigen LRH-1-reactive cytotoxic T cells.

CD8(+) T cells recognizing minor histocompatibility antigens (MiHAs) on leukemic stem and progenitor cells play a pivotal role in effective graft-versus-leukemia reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified a hematopoiesis-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene. We found that P2X5 is significantly expressed in CD34(+) leukemic subpopulations from chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. Here, we demonstrate that LRH-1-specific CD8(+) T-cell responses are frequently induced in myeloid leukemia patients following donor lymphocyte infusions. Patients with high percentages of circulating LRH-1-specific CD8(+) T cells had no or only mild graft-versus-host disease. Functional analysis showed that LRH-1-specific cytotoxic T lymphocytes (CTLs) isolated from 2 different patients efficiently target LRH-1-positive leukemic CD34(+) progenitor cells from both CML and AML patients, whereas mature CML cells are only marginally lysed due to down-regulation of P2X5. Furthermore, we observed that relative resistance to LRH-1 CTL-mediated cell death due to elevated levels of antiapoptotic XIAP could be overcome by IFN-gamma prestimulation and increased CTL-target ratios. These findings provide a rationale for use of LRH-1 as immunotherapeutic target antigen to treat residual or persisting myeloid malignancies after allogeneic SCT.

Partial T cell-depleted allogeneic stem cell transplantation following reduced-intensity conditioning creates a platform for immunotherapy with donor lymphocyte infusion and recipient dendritic cell vaccination in multiple myeloma.

Allogeneic stem cell transplantation (SCT) in multiple myeloma (MM) may induce a curative graft-versus-myeloma (GVM) effect. Major drawback in unmanipulated reduced-intensity conditioning (RIC) SCT is the risk of severe and longstanding graft-versus-host-disease (GVHD). This study demonstrates that transplantation with a partial T cell-depleted graft creates a platform for boosting GVM immunity by preemptive donor lymphocyte infusion (DLI) and recipient dendritic cell (DC) vaccination, with limited GVHD. All 20MM patients engrafted successfully. Chimerism analysis in 19 patients evaluable at 3 months revealed that 7 patients were complete donor, whereas 12 patients were mixed chimeric. Grade II acute GVHD (aGVHD) occurred in 7 patients (35%) and only 4 patients (21%) developed chronic GVHD (cGVHD). Fourteen patients received posttransplantation immunotherapy, 8 preemptive DLI, 5 patients both DLI and DC vaccination, and 1 patient DC vaccination only. DC vaccination was associated with limited toxicity, and none of these patients developed GVHD. Importantly, overall treatment-related mortality (TRM) at 1 year was low (10%). Moreover, the overall survival (OS) is 84% with median follow-up of 27 months, and none of the patients died from progressive disease. These findings illustrate that this novel approach is associated with limited GVHD and mortality, thus creating an ideal platform for adjuvant immunotherapy.

A frameshift polymorphism in P2X5 elicits an allogeneic cytotoxic T lymphocyte response associated with remission of chronic myeloid leukemia.

Minor histocompatibility antigens (mHAgs) constitute the targets of the graft-versus-leukemia response after HLA-identical allogeneic stem cell transplantation. Here, we have used genetic linkage analysis to identify a novel mHAg, designated lymphoid-restricted histocompatibility antigen-1 (LRH-1), which is encoded by the P2X5 gene and elicited an allogeneic CTL response in a patient with chronic myeloid leukemia after donor lymphocyte infusion. We demonstrate that immunogenicity for LRH-1 is due to differential protein expression in recipient and donor cells as a consequence of a homozygous frameshift polymorphism in the donor. Tetramer analysis showed that emergence of LRH-1-specific CD8+ cytotoxic T cells in peripheral blood and bone marrow correlated with complete remission of chronic myeloid leukemia. Furthermore, the restricted expression of LRH-1 in hematopoietic cells including leukemic CD34+ progenitor cells provides evidence of a role for LRH-1-specific CD8+ cytotoxic T cells in selective graft-versus-leukemia reactivity in the absence of severe graft-versus-host disease. These findings illustrate that the P2X5-encoded mHAg LRH-1 could be an attractive target for specific immunotherapy to treat hematological malignancies recurring after allogeneic stem cell transplantation.

Successful Transfer of Umbilical Cord Blood CD34+ Hematopoietic Stem and Progenitor-derived NK Cells in Older Acute Myeloid Leukemia Patients.

Purpose: Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated ex vivo from CD34+ hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units.Experimental Design: Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 106/kg body weight) after lymphodepleting chemotherapy without cytokine boosting.Results: HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1 × 104 T cells/kg and <3 × 105 B cells/kg body weight. HSPC-NK cells were well tolerated, and neither graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting being given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, which was accompanied by augmented IL15 plasma levels. Moreover, donor chimerism up to 3.5% was found in bone marrow. Interestingly, in vivo HSPC-NK cell maturation was observed, indicated by the rapid acquisition of CD16 and KIR expression, while expression of most activating receptors was sustained. Notably, 2 of 4 patients with minimal residual disease (MRD) in bone marrow before infusion became MRD negative (<0.1%), which lasted for 6 months.Conclusions: These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential "off-the-shelf" translational immunotherapy approach in AML. Clin Cancer Res; 23(15); 4107-18. ©2017 AACR.

The Aryl Hydrocarbon Receptor Antagonist StemRegenin1 Improves In Vitro Generation of Highly Functional Natural Killer Cells from CD34(+) Hematopoietic Stem and Progenitor Cells.

Early natural killer (NK)-cell repopulation after allogeneic stem cell transplantation (allo-SCT) has been associated with reduced relapse rates without an increased risk of graft-versus-host disease, indicating that donor NK cells have specific antileukemic activity. Therefore, adoptive transfer of donor NK cells is an attractive strategy to reduce relapse rates after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK-cell infusions could be administered in this setting. However, isolation of high numbers of functional NK cells from transplant donors is challenging. Hence, we developed a cytokine-based ex vivo culture protocol to generate high numbers of functional NK cells from granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) hematopoietic stem and progenitor cells (HSPCs). In this study, we demonstrate that addition of aryl hydrocarbon receptor antagonist StemRegenin1 (SR1) to our culture protocol potently enhances expansion of CD34(+) HSPCs and induces expression of NK-cell-associated transcription factors promoting NK-cell differentiation. As a result, high numbers of NK cells with an active phenotype can be generated using this culture protocol. These SR1-generated NK cells exert efficient cytolytic activity and interferon-γ production toward acute myeloid leukemia and multiple myeloma cells. Importantly, we observed that NK-cell proliferation and function are not inhibited by cyclosporin A, an immunosuppressive drug often used after allo-SCT. These findings demonstrate that SR1 can be exploited to generate high numbers of functional NK cells from G-CSF-mobilized CD34(+) HSPCs, providing great promise for effective NK-cell-based immunotherapy after allo-SCT.

Quantification of the HA-1 gene product at the RNA level; relevance for immunotherapy of hematological malignancies.

Minor histocompatibility antigens can induce cytotoxic T cells that play an important role in the graft-versus-leukemia and graft-versus-host-disease (GvHD) activity after stem cell transplantation. Minor histocompatibility antigens (mHags) with expression limited to the hematopoietic system may have a prominent role in the graft-versus-leukemia reaction. Earlier in vitro studies demonstrated that cytotoxic T cells specific for the minor histocompatibility antigen HA-1 only lysed cells of hematopoietic origin. Despite this limited expression, an HA-1 mismatch is associated with GvHD. Yet, the hematopoietic-restricted HA-1 membrane expression motivated us to develop an ex vivo HA-1-specific protocol for cellular immunotherapy of relapsed leukemia. To ensure the feasibility and safety of such cellular therapy, broad HA-1 RNA analysis is indispensable. Here we demonstrate the hematopoietic-restricted expression at the HA-1 gene transcriptional level with high RNA expression in normal and in malignant hematopoietic cells and background expression levels in nonhematopoietic cells. In tissues that showed low HA-1 RNA expression, hematopoietic cells were present as demonstrated by CD45 RNA expression analyzed in parallel. Thus, the mHag HA-1 can function as an excellent target antigen for immunotherapy of hematological malignancies with no or low risk of GvHD.

PD-1/PD-L1 interactions contribute to functional T-cell impairment in patients who relapse with cancer after allogeneic stem cell transplantation.

Tumor relapses remain a serious problem after allogeneic stem cell transplantation (alloSCT), despite the long-term persistence of minor histocompatibility antigen (MiHA)-specific memory CD8(+) T cells specific for the tumor. We hypothesized that these memory T cells may lose their function over time in transplanted patients. Here, we offer functional and mechanistic support for this hypothesis, based on immune inhibition by programmed death-1 (PD-1) expressed on MiHA-specific CD8(+) T cells and the associated role of the PD-1 ligand PD-L1 on myeloid leukemia cells, especially under inflammatory conditions. PD-L1 was highly upregulated on immature human leukemic progenitor cells, whereas costimulatory molecules such as CD80 and CD86 were not expressed. Thus, immature leukemic progenitor cells seemed to evade the immune system by inhibiting T-cell function via the PD-1/PD-L1 pathway. Blocking PD-1 signaling using human antibodies led to elevated proliferation and IFN-γ production of MiHA-specific T cells cocultured with PD-L1-expressing leukemia cells. Moreover, patients with relapsed leukemia after initial MiHA-specific T-cell responses displayed high PD-L1 expression on CD34(+) leukemia cells and increased PD-1 levels on MiHA-specific CD8(+) T cells. Importantly, blocking PD-1/PD-L1 interactions augment proliferation of MiHA-specific CD8(+) memory T cells from relapsed patients. Taken together, our findings indicate that the PD-1/PD-L pathway can be hijacked as an immune escape mechanism in hematological malignancies. Furthermore, they suggest that blocking the PD-1 immune checkpoint offers an appealing immunotherapeutic strategy following alloSCT in patients with recurrent or relapsed disease.

Bi-directional allelic recognition of the human minor histocompatibility antigen HB-1 by cytotoxic T lymphocytes.

Human minor histocompatibility antigens (mHag) are target antigens of the graft-versus-leukemia response observed after allogeneic HLA-identical stem cell transplantation. We previously defined the molecular nature of the B cell lineage-specific mHag HB-1. The CTL epitope was identified as the decamer peptide EEKRGSLHVW presented in the context of HLA-B44. The HB-1 antigen is encoded by a locus of yet unknown function on chromosome 5q32. A single nucleotide polymorphism within this locus results in an amino acid change from histidine (H) to tyrosine (Y) at position P8 within the CTL epitope. Based on genomic information, we have developed a PCR-RFLP assay to perform HB-1 typing at the DNA level. We determined that the allelic frequency for the H and Y variant is 0.79 and 0.21, respectively. From these data, we calculated that the expected recipient disparity between HLA-B44-matched sibling pairs for HB-1H is 2.8%, whereas recipient disparity for HB-1Y is expected to be 12.4%. Therefore, we addressed whether the HB-1Y peptide is reciprocally immunogenic. We revealed that both peptide variants bind equally efficient to HLA-B44 molecules and that the H/Y substitution has no influence on formation of epitope precursor peptides by 20 S proteasome-mediated degradation. More directly, CTL recognizing the naturally presented HB-1Y peptide could be generated from a HB-1H homozygous donor using peptide-pulsed dendritic cells. Using a set of synthetic structurally related peptide variants, we found that the H/Y substitution has a major impact on TCR recognition by CTL specific for either of the HB-1 allelic homologues. HB-1 is the first human mHag described that induces bi-directional allogeneic CTL responses that may contribute to a specific graft-versus-leukemia response following allogeneic stem cell transplantation.