Engineering of CD34+ progenitor-derived natural killer cells with higher-affinity CD16a for enhanced antibody-dependent cellular cytotoxicity.

BACKGROUND AIMS: Natural killer (NK) cell transfer is a promising cellular immunotherapy for cancer. Previously, we developed a robust method to generate large NK cell numbers from CD34+ hematopoietic stem and progenitor cells (HSPCs), which exhibit strong anti-tumor activity. However, since these cells express low levels of the Fc receptor CD16a in vitro, antibody-dependent cellular cytotoxicity (ADCC) by these cells is limited. To broaden clinical applicability of our HSPC-NK cells toward less NK-sensitive malignancies, we aimed to improve ADCC through CD16a transduction. METHODS: Using wildtype and S197P mutant greater-affinity (both with V158) CD16a retroviral transgenes (i.e., a cleavable and noncleavable CD16a upon stimulation), we generated CD16a HSPC-transduced NK cells, with CD34+ cells isolated from umbilical cord blood (UCB) or peripheral blood after G-CSF stem cell mobilization (MPB). CD16a expressing NK cells were enriched using flow cytometry-based cell sorting. Subsequently, phenotypic analyses and functional assays were performed to investigate natural cytotoxicity and ADCC activity. RESULTS: Mean transduction efficiency was 34% for UCB-derived HSPCs and 20% for MPB-derived HSPCs, which was enriched by flow cytometry-based cell sorting to >90% for both conditions. Expression of the transgene remained stable during the entire NK expansion cell generation process. Proliferation and differentiation of HSPCs were not hampered by the transduction process, resulting in effectively differentiated CD56+ NK cells after 5 weeks. Activation of the HSPC-derived NK cells resulted in significant shedding of wildtype CD16a transcribed from the endogenous gene, but not of the noncleavable mutant CD16a protein expressed from the transduced construct. The mean increase of CD107+IFNγ+ expressing NK cells after inducing ADCC was tenfold in enriched noncleavable CD16a HSPC-NK cells. Killing capacity of CD16a-transduced NK cells was significantly improved after addition of a tumor-targeting antibody in tumor cell lines and primary B-cell leukemia and lymphoma cells compared to unmodified HSPC-NK cells. CONCLUSIONS: Together, these data demonstrate that the applicability of adoptive NK cell immunotherapy may be broadened to less NK-sensitive malignancies by upregulation of CD16a expression in combination with the use of tumor-targeting monoclonal antibodies.

AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival.

BACKGROUND: AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets. METHODS: VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale. RESULTS: AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP. CONCLUSION: Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse.

Human CD34+-derived complete plasmacytoid and conventional dendritic cell vaccine effectively induces antigen-specific CD8+ T cell and NK cell responses in vitro and in vivo.

Allogeneic stem cell transplantation (alloSCT) can be curative for hemato-oncology patients due to effective graft-versus-tumor immunity. However, relapse remains the major cause of treatment failure, emphasizing the need for adjuvant immunotherapies. In this regard, post-transplantation dendritic cell (DC) vaccination is a highly interesting strategy to boost graft-versus-tumor responses. Previously, we developed a clinically applicable protocol for simultaneous large-scale generation of end-stage blood DC subsets from donor-derived CD34+ stem cells, including conventional type 1 and 2 DCs (cDC1s and cDC2s), and plasmacytoid DCs (pDCs). In addition, the total cultured end-product (DC-complete vaccine), also contains non-end-stage-DCs (i.e. non-DCs). In this study, we aimed to dissect the phenotypic identity of these non-DCs and their potential immune modulatory functions on the potency of cDCs and pDCs in stimulating tumor-reactive CD8+ T and NK cell responses, in order to obtain rationale for clinical translation of our DC-complete vaccine. The non-DC compartment was heterogeneous and comprised of myeloid progenitors and (immature) granulocyte- and monocyte-like cells. Importantly, non-DCs potentiated toll-like receptor-induced DC maturation, as reflected by increased expression of co-stimulatory molecules and enhanced cDC-derived IL-12 and pDC-derived IFN-α production. Additionally, antigen-specific CD8+ T cells effectively expanded upon DC-complete vaccination in vitro and in vivo. This effect was strongly augmented by non-DCs in an antigen-independent manner. Moreover, non-DCs did not impair in vitro DC-mediated NK cell activation, degranulation nor cytotoxicity. Notably, in vivo i.p. DC-complete vaccination activated i.v. injected NK cells. Together, these data demonstrate that the non-DC compartment potentiates DC-mediated activation and expansion of antigen-specific CD8+ T cells and do not impair NK cell responses in vitro and in vivo. This underscores the rationale for further clinical translation of our CD34+-derived DC-complete vaccine in hemato-oncology patients post alloSCT.

CD34+ progenitor-derived NK cell and gemcitabine combination therapy increases killing of ovarian cancer cells in NOD/SCID/IL2Rgnull mice.

Combining natural killer (NK) cell adoptive transfer with tumor-sensitizing chemotherapy is an attractive approach against recurrent ovarian cancer (OC), as OC is sensitive to NK cell-mediated immunity. Previously, we showed that CD34+ hematopoietic progenitor cell (HPC)-derived NK cells can kill OC cells in vitro and inhibit OC tumor growth in mice. Here, we investigated the potential of HPC-NK cell therapy combined with chemotherapeutic gemcitabine (used in recurrent OC patients) against OC. We examined the phenotypical, functional, and cytotoxic effects of gemcitabine on HPC-NK cells and/or OC cells in vitro and in OC-bearing mice. To this end, we treated OC cells and/or HPC-NK cells with or without gemcitabine and analyzed the phenotype, cytokine production, and anti-tumor reactivity. We found that gemcitabine did not affect the phenotype and functionality of HPC-NK cells, while on OC cells expression of NK cell activating ligands and death receptors was upregulated. Although gemcitabine pre-treatment of OC cells did not improve the functionality of HPC-NK cells, importantly, HPC-NK cells and gemcitabine additively killed OC cells in vitro. Similarly, combined HPC-NK cell and gemcitabine treatment additively decreased tumor growth in OC-bearing mice. Collectively, our results indicate that combination therapy of HPC-NK cells and gemcitabine results in augmented OC killing in vitro and in vivo. This provides a rationale for exploring this therapeutic strategy in patients with recurrent OC.

Clinically applicable CD34+-derived blood dendritic cell subsets exhibit key subset-specific features and potently boost anti-tumor T and NK cell responses.

Allogeneic stem cell transplantation (alloSCT), following induction chemotherapy, can be curative for hemato-oncology patients due to powerful graft-versus-tumor immunity. However, disease recurrence remains the major cause of treatment failure, emphasizing the need for potent adjuvant immunotherapy. In this regard, dendritic cell (DC) vaccination is highly attractive, as DCs are the key orchestrators of innate and adaptive immunity. Natural DC subsets are postulated to be more powerful compared with monocyte-derived DCs, due to their unique functional properties and cross-talk capacity. Yet, obtaining sufficient numbers of natural DCs, particularly type 1 conventional DCs (cDC1s), is challenging due to low frequencies in human blood. We developed a clinically applicable culture protocol using donor-derived G-CSF mobilized CD34+ hematopoietic progenitor cells (HPCs) for simultaneous generation of high numbers of cDC1s, cDC2s and plasmacytoid DCs (pDCs). Transcriptomic analyses demonstrated that these ex vivo-generated DCs highly resemble their in vivo blood counterparts. In more detail, we demonstrated that the CD141+CLEG9A+ cDC1 subset exhibited key features of in vivo cDC1s, reflected by high expression of co-stimulatory molecules and release of IL-12p70 and TNF-α. Furthermore, cDC1s efficiently primed alloreactive T cells, potently cross-presented long-peptides and boosted expansion of minor histocompatibility antigen-experienced T cells. Moreover, they strongly enhanced NK cell activation, degranulation and anti-leukemic reactivity. Together, we developed a robust culture protocol to generate highly functional blood DC subsets for in vivo application as tailored adjuvant immunotherapy to boost innate and adaptive anti-tumor immunity in alloSCT patients.

PD-L1 siRNA-mediated silencing in acute myeloid leukemia enhances anti-leukemic T cell reactivity.

Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients' AML cells. Importantly, WT1-antigen T cell receptor+ PD-1+ 2D3 cells showed increased activation toward PD-L1 silenced WT1+ AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-γ production of minor histocompatibility antigen-specific CD8+ T cells. Notably, PD-L1 silencing was equally effective as PD-1 antibody blockade. Together, our study demonstrates that PD-L1 silencing may be an effective strategy to augment AML immune-susceptibility. This provides rationale for further development of targeted approaches to locally interfere with immune escape mechanisms in AML, thereby minimizing severe toxicity. In combination with alloSCT and/or adoptive T cell transfer, this strategy could be very appealing to boost graft-versus-leukemia immunity and improve outcome in AML patients.

Cell composition and expansion strategy can reduce the beneficial effect of AKT-inhibition on functionality of CD8+ T cells.

AKT-inhibition is a promising approach to improve T cell therapies; however, its effect on CD4+ T cells is insufficiently explored. Previously, we and others showed that AKT-inhibition during ex vivo CD8+ T cell expansion facilitates the generation of polyfunctional T cells with stem cell memory-like traits. However, most therapeutic T cell products are generated from lymphocytes, containing CD4+ T cells that can affect CD8+ T cells dependent on the Th-subset. Here, we investigated the effect of AKT-inhibition on CD4+ T cells, during separate as well as total T cell expansions. Interestingly, ex vivo AKT-inhibition preserved the early memory phenotype of CD4+ T cells based on higher CD62L, CXCR4 and CCR7 expression. However, in the presence of AKT-inhibition, Th-differentiation was skewed toward more Th2-associated at the expense of Th1-associated cells. Importantly, the favorable effect of AKT-inhibition on the functionality of CD8+ T cells drastically diminished in the presence of CD4+ T cells. Moreover, also the expansion method influenced the effect of AKT-inhibition on CD8+ T cells. These findings indicate that the effect of AKT-inhibition on CD8+ T cells is dependent on cell composition and expansion strategy, where presence of CD4+ T cells as well as polyclonal stimulation impede the favorable effect of AKT-inhibition.

Comprehensive Phenotyping of T Cells Using Flow Cytometry.

The T cell compartment can form a powerful defense against extrinsic (e.g., pathogens) and intrinsic danger (e.g., malignant cells). At the same time, specific subsets of T cells control this process to keep the immune system in check and prevent autoimmunity. A wide variety in T cell functionalities exists, which is dependent on the differentiation and maturation state of the T cells. In this review, we report an overview for the identification of CD4+ T-αß cells (T-helper (Th)1, Th2, Th9, Th17, Th22, and CD4+ regulatory T cells), CD8+ T-αß cells (cytotoxic T lymphocyte (Tc)1, Tc2, Tc9, Tc17, and CD8+ regulatory T cells), and their additional effector memory status (naïve, stem cell memory, central memory, effector memory, and effector) using flow cytometry. These different subsets can be discriminated based on selective extracellular markers, in combination with intracellular transcription factor and/or cytokine stainings. Additionally, identification of very small subsets, including antigen-specific T cells, and important technical considerations of flow cytometry are discussed. Together, this overview can be used for comprehensive phenotyping of a T cell subset of interest. © 2019 International Society for Advancement of Cytometry.

Ex vivo AKT-inhibition facilitates generation of polyfunctional stem cell memory-like CD8+ T cells for adoptive immunotherapy.

Adoptive T cell therapy has shown clinical potential for patients with cancer, though effective treatment is dependent on longevity and potency of the exploited tumor-reactive T cells. Previously, we showed that ex vivo inhibition of AKT using the research compound Akt-inhibitor VIII retained differentiation and improved functionality of minor histocompatibility antigen (MiHA)-specific CD8+ T cells. Here, we compared a panel of clinically applicable AKT-inhibitors with an allosteric or adenosine triphosphate-competitive mode of action. We analyzed phenotype, functionality, metabolism and transcriptome of AKT-inhibited CD8+ T cells using different T cell activation models. Most inhibitors facilitated T cell expansion while preserving an early memory phenotype, reflected by maintenance of CD62L, CCR7 and CXCR4 expression. Moreover, transcriptome profiling revealed that AKT-inhibited CD8+ T cells clustered closely to naturally occurring stem cell-memory CD8+ T cells, while control T cells resembled effector-memory T cells. Interestingly, AKT-inhibited CD8+ T cells showed enrichment of hypoxia-associated genes, which was consistent with enhanced glycolytic function. Notably, AKT-inhibition during MiHA-specific CD8+ T cell priming uncoupled preservation of early memory differentiation from ex vivo expansion. Furthermore, AKT-inhibited MiHA-specific CD8+ T cells showed increased polyfunctionality with co-secretion of IFN-γ and IL-2 upon antigen recall. Together, these data demonstrate that AKT-inhibitors with different modality of action promote the ex vivo generation of stem cell memory-like CD8+ T cells with a unique metabolic profile and retained polyfunctionality. Akt-inhibitor VIII and GDC-0068 outperformed other inhibitors, and are therefore promising candidates for ex vivo generation of superior tumor-reactive T cells for adoptive immunotherapy in cancer patients.

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.

Combined IL-15 and IL-12 drives the generation of CD34+-derived natural killer cells with superior maturation and alloreactivity potential following adoptive transfer.

Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against cancer, including acute myeloid leukemia (AML). Previously, we reported a cytokine-based culture method for the generation of NK cell products with high cell number and purity. In this system, CD34+ hematopoietic progenitor cells (HPC) were expanded and differentiated into NK cells under stroma-free conditions in the presence of IL-15 and IL-2. We show that combining IL-15 with IL-12 drives the generation of more mature and highly functional NK cells. In particular, replacement of IL-2 by IL-12 enhanced the cytolytic activity and IFNγ production of HPC-NK cells toward cultured and primary AML cells in vitro, and improved antileukemic responses in NOD/SCID-IL2Rγnull (NSG) mice bearing human AML cells. Phenotypically, IL-12 increased the frequency of HPC-NK cells expressing NKG2A and killer immunoglobulin-like receptor (KIR), which were more responsive to target cell stimulation. In addition, NK15/12 cell products demonstrated superior maturation potential, resulting in >70% positivity for CD16 and/or KIR within 2 weeks after infusion into NSG mice. We predict that higher functionality and faster in vivo maturation will favor HPC-NK cell alloreactivity toward malignant cells in patients, making this cytokine combination an attractive strategy to generate clinical HPC-NK cell products for cancer adoptive immunotherapy.

Time to Akt: Superior tumor-reactive T cells for adoptive immunotherapy.

T cells are crucial players in the protection against cancer, and can be used in adoptive cell therapy to prevent or treat relapse. However, their state of differentiation determines their effectiveness, with early memory cells being the most favorable. Here, we discuss restraining of differentiation to engineer the ultimate tumor-reactive T cell.

siRNA silencing of PD-1 ligands on dendritic cell vaccines boosts the expansion of minor histocompatibility antigen-specific CD8(+) T cells in NOD/SCID/IL2Rg(null) mice.

Allogeneic stem cell transplantation (allo-SCT) can be a curative therapy for patients suffering from hematological malignancies. The therapeutic efficacy is based on donor-derived CD8(+) T cells that recognize minor histocompatibility antigens (MiHAs) expressed by patient's tumor cells. However, these responses are not always sufficient, and persistence and recurrence of the malignant disease are often observed. Therefore, application of additive therapy targeting hematopoietic-restricted MiHAs is essential. Adoptive transfer of MiHA-specific CD8(+) T cells in combination with dendritic cell (DC) vaccination could be a promising strategy. Though effects of DC vaccination in anti-cancer therapy have been demonstrated, improvement in DC vaccination therapy is needed, as clinical responses are limited. In this study, we investigated the potency of program death ligand (PD-L) 1 and 2 silenced DC vaccines for ex vivo priming and in vivo boosting of MiHA-specific CD8(+) T cell responses. Co-culturing CD8(+) T cells with MiHA-loaded DCs resulted in priming and expansion of functional MiHA-specific CD8(+) T cells from the naive repertoire, which was augmented upon silencing of PD-L1 and PD-L2. Furthermore, DC vaccination supported and expanded adoptively transferred antigen-specific CD8(+) T cells in vivo. Importantly, the use of PD-L silenced DCs improved boosting and further expansion of ex vivo primed MiHA-specific CD8(+) T cells in immunodeficient mice. In conclusion, adoptive transfer of ex vivo primed MiHA-specific CD8(+) T cells in combination with PD-L silenced DC vaccination, targeting MiHAs restricted to the hematopoietic system, is an interesting approach to boost GVT immunity in allo-SCT patients and thereby prevent relapse.

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.

Ex vivo generation of interstitial and Langerhans cell-like dendritic cell subset-based vaccines for hematological malignancies.

Autologous, patient-specific, monocyte-derived dendritic cell (MoDC) vaccines have been successfully applied in the clinical studies so far. However, the routine application of this strategy has been hampered by the difficulties in generating sufficient numbers of DC and the poor DC vaccine quality because of pathology or prior treatment received by the patients. The immunotherapeutic potential of other subsets of DC has not been thoroughly investigated because of their rarity in tissues and difficulties associated with their ex vivo generation. The high expansion and differentiation potential of CD34 hematopoietic progenitor cells (HPC), isolated from umbilical cord blood (UCB), into different DC subsets make them an attractive alternative DC source for cancer immunotherapy. Therefore, the aim of this study was to generate a large number of different DC subsets from CD34 HPC and evaluate their functionality in comparison with MoDC. Our culture protocol generated a clinically relevant number of mature CD1a myeloid DC and CD207 Langerhans cells (LC)-like DC subsets from CD34 HPC with >95% purity. Both DC subsets exhibited a cytokine profile that favors cytotoxic T-cell responses. Furthermore, UCB-DC and UCB-LC demonstrated superior induction of proliferation of both allogeneic as well as viral antigen-specific CD8 T cells, both in vitro and in vivo. Additional studies revealed that UCC-DC and UCB-LC can efficiently expand minor histocompatibility antigen (MiHA) HA-1-specific cytotoxic T cells in the peripheral blood of leukemia patients and prime MiHA HA-1-specific and HA-2-specific cytotoxic T cells in vitro. These preclinical findings support the pharmaceutical development of the described culture protocol for clinical evaluation.

Targeting the IL17 pathway for the prevention of graft-versus-host disease.

Graft-versus-host disease (GVHD) is still a major complication of allogeneic stem cell transplantation (allo-SCT). The pathophysiology of GVHD is a multistep process initiated by tissue damage and proinflammatory cytokine cascades induced by the pretransplantation conditioning therapy. This eventually results in Th1-driven tissue damage. However, increasing evidence indicates the involvement of IL17-producing T cells in GVHD pathogenesis. Both CD4(+) and CD8(+) IL17-producing T cells are suspected of initiating the Th1 response and aggravating tissue inflammation, resulting in full-blown GVHD. In this review, we discuss the involvement of IL17-producing T cells in GVHD and the factors involved in their expansion, differentiation, and activation. Different dendritic cell (DC) subsets, such as plasmacytoid DCs and DC NK lectin group receptor 1(+) myeloid DCs have the capability to stimulate Th/Tc17 responses through the release of cytokines. Pivotal cytokines include IL1ß, IL6, IL23, and TGFß, which are known to drive differentiation and expansion of IL17-producing T cells, and these cytokines are highly elevated in patients after allo-SCT. Potent activators of these DC subsets are motifs that are released upon tissue damage and microbial exposure during allo-SCT. These motifs aggravate the Th/Tc17 response via the activation of various pathogen recognition receptors, thereby initiating and perpetuating GVHD. A more comprehensive understanding of the factors and DC subsets driving the IL17 pathway will result in developing and testing novel therapeutic approaches for the prevention of GVHD.

Natural killer cells generated from cord blood hematopoietic progenitor cells efficiently target bone marrow-residing human leukemia cells in NOD/SCID/IL2Rg(null) mice.

Natural killer (NK) cell-based adoptive immunotherapy is an attractive adjuvant treatment option for patients with acute myeloid leukemia. Recently, we reported a clinical-grade, cytokine-based culture method for the generation of NK cells from umbilical cord blood (UCB) CD34⁺ hematopoietic progenitor cells with high yield, purity and in vitro functionality. The present study was designed to evaluate the in vivo anti-leukemic potential of UCB-NK cells generated with our GMP-compliant culture system in terms of biodistribution, survival and cytolytic activity following adoptive transfer in immunodeficient NOD/SCID/IL2Rg(null) mice. Using single photon emission computed tomography, we first demonstrated active migration of UCB-NK cells to bone marrow, spleen and liver within 24 h after infusion. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Thereafter, we showed that low dose IL-15 mediates efficient survival, expansion and maturation of UCB-NK cells in vivo. Most importantly, we demonstrate that a single UCB-NK cell infusion combined with supportive IL-15 administration efficiently inhibited growth of human leukemia cells implanted in the femur of mice, resulting in significant prolongation of mice survival. These preclinical studies strongly support the therapeutic potential of ex vivo-generated UCB-NK cells in the treatment of myeloid leukemia after immunosuppressive chemotherapy.

Decreased levels of circulating IL17-producing CD161+CCR6+ T cells are associated with graft-versus-host disease after allogeneic stem cell transplantation.

The C-type lectin-like receptor CD161 is a well-established marker for human IL17-producing T cells, which have been implicated to contribute to the development of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT). In this study, we analyzed CD161(+) T cell recovery, their functional properties and association with GVHD occurrence in allo-SCT recipients. While CD161(+)CD4(+) T cells steadily recovered, CD161(hi)CD8(+) T cell numbers declined during tapering of Cyclosporine A (CsA), which can be explained by their initial growth advantage over CD161(neg/low)CD8(+) T cells due to ABCB1-mediated CsA efflux. Interestingly, occurrence of acute and chronic GVHD was significantly correlated with decreased levels of circulating CD161(+)CD4(+) as well as CD161(hi)CD8(+) T cells. In addition, these subsets from transplanted patients secreted high levels of IFNγ and IL17. Moreover, we found that CCR6 co-expression by CD161(+) T cells mediated specific migration towards CCL20, which was expressed in GVHD biopsies. Finally, we demonstrated that CCR6(+) T cells indeed were present in these CCL20(+) GVHD-affected tissues. In conclusion, we showed that functional CD161(+)CCR6(+) co-expressing T cells disappear from the circulation and home to GVHD-affected tissue sites. These findings support the hypothesis that CCR6(+)CD161-expressing T cells may be involved in the immune pathology of GVHD following their CCL20-dependent recruitment into affected tissues.

Polymorphisms in CCR6 are associated with chronic graft-versus-host disease and invasive fungal disease in matched-related hematopoietic stem cell transplantation.

Graft-versus-host disease (GVHD) and fungal infections are frequent complications after allogeneic hematopoietic stem cell transplantation (HSCT). Single nucleotide polymorphisms (SNPs) in genes of the immune system can influence the inflammatory cascade and T cell-driven alloimmune reactions after HSCT, and thus increasing the incidence of GVHD and infectious complications. Here, we investigated the effect of SNPs in IL-23R and CCR6 on posttransplantation outcome in 161 recipients of partially T cell-depleted HSCT. Remarkably, IL-23R SNPs were not associated with clinical outcome, but we found that disparities in the CCR6 tagSNP rs2301436 and SNP rs3093023 are independently associated with the occurrence of chronic GVHD (cGVHD) and invasive fungal disease. In multivariate analysis, patients receiving a transplant from a homozygous rs2301436 G allele donor showed less cGVHD (odds ratio [OR]: 0.16; P = .002), as was the case for a homozygous donor rs3093023 G allele (OR: 0.24; P = .005). In parallel, the GG genotype at rs2301436 in donors was associated with a higher incidence of invasive fungal disease at day 100 after HSCT (OR: 3.59; P = .008). This study shows that CCR6 SNPs can be used to predict clinical outcome, and that polymorphisms in the CCR6 gene may influence T cell-mediated immune reactions after HSCT.