Generation of human ILC3 from allogeneic and autologous CD34+ hematopoietic progenitors toward adoptive transfer.

Type 3 innate lymphoid cells (ILC3) are important in tissue homeostasis. In the gut, ILC3 repair damaged epithelium and suppress inflammation. In allogeneic hematopoietic cell transplantation (HCT), ILC3 protect against graft-versus-host disease (GvHD), most likely by restoring tissue damage and preventing inflammation. We hypothesize that supplementing HCT grafts with interleukin-22 (IL-22)-producing ILC3 may prevent acute GvHD. We therefore explored ex vivo generation of human IL-22-producing ILC3 from hematopoietic stem and progenitor cells (HSPC) obtained from adult, neonatal and fetal sources. We established a stroma-free system culturing human cord blood-derived CD34+ HSPC with successive cytokine mixes for 5 weeks. We analyzed the presence of phenotypically defined ILC, their viability, proliferation and IL-22 production (after stimulation) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We found that the addition of recombinant human IL-15 and the enhancer of zeste homolog 1/2 inhibitor UNC1999 promoted ILC3 generation. Similar results were demonstrated when UNC1999 was added to CD34+ HSPC derived from healthy adult granulocyte colony-stimulating factor mobilized peripheral blood and bone marrow, but not fetal liver. UNC1999 did not negatively impact IL-22 production in any of the HSPC sources. Finally, we observed that autologous HSPC mobilized from the blood of adults with hematological malignancies also developed into ILC3, albeit with a significantly lower capacity. Together, we developed a stroma-free protocol to generate large quantities of IL-22-producing ILC3 from healthy adult human HSPC that can be applied for adoptive transfer to prevent GvHD after allogeneic HCT.

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.

Current challenges in cell and gene therapy: a joint view from the European Committee of the International Society for Cell & Gene Therapy (ISCT) and the European Society for Blood and Marrow Transplantation (EBMT).

Cell and gene therapy poses evolving challenges. The current article summarizes the discussions held by European Regional Committee of the International Society for Cell & Gene Therapy and the European Society for Blood and Marrow Transplantation (EBMT) on the current challenges in this field, focusing on the European setting. This article emphasizes the imperative assessment of real-world cell and gene therapy activity, advocating for expanded registries beyond hematopoietic transplantation and chimeric antigen receptor-T-cell therapy. Accreditation's role in ensuring standardized procedures, as exemplified by JACIE (The Joint Accreditation Committee of ISCT-Europe and EBMT), is crucial for safety. Access to commercial products and reimbursement variations among countries underscore the need for uniform access to advanced therapy medical products (ATMPs). Academic product development and point-of-care manufacturing face barriers to patient access. Hospital Exemption's potential, demonstrated by some initial experiences, may increase patient accessibility in individual situations. Regulatory challenges, including the ongoing European ATMPs legislation review, necessitate standardized criteria for Hospital Exemption and mandatory reporting within registries. Efforts to combat unproven therapies and fraud involve collaboration between scientific societies, regulatory bodies and patient groups. Finally, is important to highlight the vital role of education and workforce development in meeting the escalating demand for specialized professionals in the ATMP field. Collaboration among scientific societies, academic institutions, industry, regulatory bodies and patient groups is crucial for overcoming all these challenges to increase gene and cell therapy activity in Europe.

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.

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.

CXCR4, but not CXCR3, drives CD8+ T-cell entry into and migration through the murine bone marrow.

The BM serves as a blood-forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T-cell migration to and localization inside the BM. As BM accumulates many CXCR3-expressing memory CD8+ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8+ T cells in BM, is critically important for homing of all CD8+ T-cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8+ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM-1+VCAM-1+BP-1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL-7 and IL-15. We therefore conclude that CXCR4 is not only crucial for entry of CD8+ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.

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.

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.

What does cell therapy manufacturing cost? A framework and methodology to facilitate academic and other small-scale cell therapy manufacturing costings.

BACKGROUND AIMS: Recent technical and clinical advances with cell-based therapies (CBTs) hold great promise in the treatment of patients with rare diseases and those with high unmet medical need. Currently the majority of CBTs are developed and manufactured in specialized academic facilities. Due to small scale, unique characteristics and specific supply chain, CBT manufacturing is considered costly compared to more conventional medicinal products. As a result, biomedical researchers and clinicians are increasingly faced with cost considerations in CBT development. The objective of this research was to develop a costing framework and methodology for academic and other small-scale facilities that manufacture cell-based therapies. METHODS: We conducted an international multi-center costing study in four facilities in Europe using eight CBTs as case studies. This study includes costs from cell or tissue procurement to release of final product for clinical use. First, via interviews with research scientists, clinicians, biomedical scientists, pharmacists and technicians, we designed a high-level costing framework. Next, we developed a more detailed uniform methodology to allocate cost items. Costs were divided into steps (tissue procurement, manufacturing and fill-finish). The steps were each subdivided into cost categories (materials, equipment, personnel and facility), and each category was broken down into facility running (fixed) costs and operational (variable) costs. The methodology was tested via the case studies and validated in developer interviews. Costs are expressed in 2018 euros (€). RESULTS: The framework and methodology were applicable across facilities and proved sensitive to differences in product and facility characteristics. Case study cost estimates ranged between €23 033 and €190 799 Euros per batch, with batch yield varying between 1 and 88 doses. The cost estimations revealed hidden costs to developers and provided insights into cost drivers to help design manufacturing best practices. CONCLUSIONS: This framework and methodology provide step-by-step guidance to estimate manufacturing costs specifically for cell-based therapies manufactured in academic and other small-scale enterprises. The framework and methodology can be used to inform and plan cost-conscious strategies for CBTs.

Harnessing natural killer cells for the treatment of ovarian cancer.

INTRODUCTION: Adoptive cellular immunotherapy could be an interesting new treatment option for ovarian carcinoma (OC), as research has demonstrated that OC is an immunogenic disease. In particular, natural killer (NK) cells have attracted attention due to their ability to kill tumor cells without prior sensitization. The therapeutic value of allogeneic NK cells has been first observed in hematological cancers and is increasingly being explored in solid tumors. METHODS: To substantiate the rationale for NK cell therapy in OC we performed a literature search in the Pubmed database and in the international trial register clinicaltrials.gov with attention for the effect of OC on NK cell function, the effect of current treatment on NK cell biology and the evidence on the therapeutic value of NK cell therapy against OC. RESULTS: In six clinical trials only 31 OC patients have been reported that received NK cell adoptive transfer. The majority of patients reached stable disease after NK cell therapy, with a mild pattern of side effects. In patients who received repeated infusions, more complete responses are described. All reported studies investigated the intravenous infusion of NK cells. Whereas the studies that are currently recruiting, investigate intraperitoneal infusion of allogeneic NK cells. CONCLUSION: In this review the pre-clinical evidence and current trials on NK cell immunotherapy in OC patients are summarized. Furthermore, challenges that have to be overcome for NK cell adoptive therapy to have a significant impact on disease outcome are discussed.

Phase I/II Trial of a Combination of Anti-CD3/CD7 Immunotoxins for Steroid-Refractory Acute Graft-versus-Host Disease.

Effective therapies for treating patients with steroid-refractory acute graft-versus-host-disease (SR-aGVHD), particularly strategies that reduce the duration of immunosuppression following remission, are urgently needed. The investigated immunotoxin combination consists of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT), which induces in vivo depletion of T cells and natural killer (NK) cells and suppresses T cell receptor activation. We conducted a phase I/II trial to examine the safety and efficacy of CD3/CD7-IT in 20 patients with SR-aGVHD; 17 of these patients (85%) had severe SR-aGVHD, and all 20 patients had visceral organ involvement, including 18 (90%) with gastrointestinal (GI) involvement and 5 (25%) with liver involvement. A validated 2-biomarker algorithm classified the majority of patients (11 of 20) as high risk. On day 28 after the start of CD3/CD7-IT therapy, the overall response rate was 60% (12 of 20), with 10 patients (50%) achieving a complete response. The 6-month overall survival rate was 60% (12 of 20), including 64% (7 of 11) classified as high risk by biomarkers. The 1-week course of treatment with CD3/CD7-IT caused profound but transient depletion of T cells and NK cells, followed by rapid recovery of the immune system with a diverse TCR Vß repertoire, and preservation of Epstein-Barr virus- and cytomegalovirus-specific T cell clones. Furthermore, our results indicate that CD3/CD7-IT appeared to be safe and well tolerated, with a relatively low prevalence of manageable and reversible adverse events, primarily worsening of hypoalbuminemia, microangiopathy, and thrombocytopenia. These encouraging results suggest that CD3/CD7-IT may improve patient outcomes in patients with SR-aGVHD.

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.

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.

Immune checkpoint molecules in acute myeloid leukaemia: managing the double-edged sword.

New immunotherapeutic interventions have revolutionized cancer treatment. The immune responsiveness of acute myeloid leukaemia (AML) was first demonstrated by allogeneic stem cell transplantation. In addition, milder immunotherapeutic approaches are exploited. However, the long-term efficacy of these therapies is hampered by various immune resistance and editing mechanisms. In this regard, co-inhibitory signalling pathways have been shown to play a crucial role. Via up-regulation of inhibitory checkpoints, tumour-reactive T cell and Natural Killer cell responses can be strongly impeded. Accordingly, the introduction of checkpoint inhibitors targeting CTLA-4 (CTLA4) and PD-1 (PDCD1, CD279)/PD-L1 (CD274, PDCD1LG1) accomplished a breakthrough in cancer treatment, with impressive clinical responses. Numerous new co-inhibitory players and novel combination therapies are currently investigated for their potential to boost anti-tumour immunity and improve survival of cancer patients. Although the challenge here remains to avoid severe systemic toxicity. This review addresses the involvement of co-inhibitory signalling in AML immune evasion and discusses the opportunities for checkpoint blockers in AML treatment.

CLEC12A-Mediated Antigen Uptake and Cross-Presentation by Human Dendritic Cell Subsets Efficiently Boost Tumor-Reactive T Cell Responses.

Potent immunotherapies are urgently needed to boost antitumor immunity and control disease in cancer patients. As dendritic cells (DCs) are the most powerful APCs, they are an attractive means to reinvigorate T cell responses. An appealing strategy to use the effective Ag processing and presentation machinery, T cell stimulation and cross-talk capacity of natural DC subsets is in vivo tumor Ag delivery. In this context, endocytic C-type lectin receptors are attractive targeting molecules. In this study, we investigated whether CLEC12A efficiently delivers tumor Ags into human DC subsets, facilitating effective induction of CD4(+) and CD8(+) T cell responses. We confirmed that CLEC12A is selectively expressed by myeloid cells, including the myeloid DC subset (mDCs) and the plasmacytoid DC subset (pDCs). Moreover, we demonstrated that these DC subsets efficiently internalize CLEC12A, whereupon it quickly translocates to the early endosomes and subsequently routes to the lysosomes. Notably, CLEC12A Ab targeting did not negatively affect DC maturation or function. Furthermore, CLEC12A-mediated delivery of keyhole limpet hemocyanin resulted in enhanced proliferation and cytokine secretion by keyhole limpet hemocyanin-experienced CD4(+) T cells. Most importantly, CLEC12A-targeted delivery of HA-1 long peptide resulted in efficient Ag cross-presentation by mDCs and pDCs, leading to strong ex vivo activation of HA-1-specific CD8(+) T cells of patients after allogeneic stem cell transplantation. Collectively, these data indicate that CLEC12A is an effective new candidate with great potential for in vivo Ag delivery into mDCs and pDCs, thereby using the specialized functions and cross-talk capacity of these DC subsets to boost tumor-reactive T cell immunity in cancer patients.

Addition of 10-Day Decitabine to Fludarabine/Total Body Irradiation Conditioning is Feasible and Induces Tumor-Associated Antigen-Specific T Cell Responses.

Allogeneic hematopoietic cell transplantation (HCT) offers the possibility of curative therapy for patients with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and acute myelogenous leukemia (AML). However, post-HCT relapse remains a major problem, particularly in patients with high-risk cytogenetics and in patients who cannot tolerate consolidation chemotherapy (eg, due to previous toxicity). We assessed the toxicity and efficacy of 10-day decitabine (Dec), fludarabine (Flu), and 2 Gy total body irradiation (TBI) as a new conditioning regimen for allogeneic HCT in patients with MDS, CMML, or AML. Thirty patients were enrolled, including 11 with MDS, 2 with CMML, and 17 with AML. Patients received 20 mg/m(2)/day Dec on days -11 to -2, 30 mg/m(2)/day Flu on days -4 to -2, and 2 Gy TBI on day -1, followed by infusion of a donor stem cell graft on day 0. Postgrafting immunosuppression consisted of cyclosporin A and mycophenolate mofetil. At a median follow-up of 443 days, the overall survival was 53%, relapse incidence was 27%, and nonrelapse mortality was 27%. The incidence of severe acute (grade III/IV) graft-versus-host disease (GVHD) was 27%, and that of (predominantly mild) chronic GVHD was 60%. Immunomonitoring studies revealed that specific CD8(+) T cell responses against epigenetically silenced tumor-associated antigens (TAAs), including cancer-testis antigens (MAGE-A1/A2/A3 and PRAME) and RHAMM, occurred more frequently in patients who had received Dec/Flu/TBI conditioning (8 of 11 patients) compared with a control group of patients who had received only Flu/TBI conditioning (2 of 9 patients). In summary, Dec/Flu/TBI conditioning proved feasible and effective and enhanced the induction of TAA-reactive CD8(+) T cell responses in vivo, which may contribute to disease control post-transplantation.

The magnitude of cytokine production by stimulated CD56+ cells is associated with early stages of systemic sclerosis.

Immune activation is a hallmark of systemic sclerosis (SSc). However, the immunological alterations that occur in preclinical and non-fibrotic SSc and that differentiate these subjects from those with primary Raynaud's phenomenon (PRP) or healthy controls (HC) are poorly defined. We isolated CD56+ (NK/NKT-like) cells from HC, patients with PRP, early SSc (EaSSc) and definite SSc without skin or lung fibrosis. Cytokine production upon different activating stimuli was measured via a multiplex immuno assay. Clearly discriminative patterns among the different stages of SSc were most markedly observed after TLR1/2 stimulation, with increased IL-6, TNF-α and MIP-1α/CCL3 production in definite SSc patients as compared to HC and/or PRP. Initial alterations were observed in EaSSc patients with an intermediate secretion pattern between HC/PRP and definite SSc. CD56+ cells from patients at different stages of SSc differentially respond to TLR stimulation, highlighting the relevance of natural immunity in the developmental and pre-fibrotic SSc.

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.

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.

Umbilical cord blood-derived cellular products for cancer immunotherapy.

Although the vast majority of experience with umbilical cord blood (CB) centers on hematopoietic reconstitution, a recent surge in the knowledge of CB cell subpopulations as well as advances in ex vivo culture technology have expanded the potential of this rich resource. Because CB has the capacity to generate the entire hematopoietic system, we now have a new source for natural killer, dendritic and T cells for therapeutic use against malignancies. This Review will focus on cellular immunotherapies derived from CB. Expansion techniques, ongoing clinical trials and future directions for this new dimension of CB application are also discussed.