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Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793.

Background Left ventricular hypertrophy and heart failure with preserved ejection fraction (HFpEF) are primary manifestations of the cardiorenal syndrome in patients with chronic kidney disease (CKD). Therapies that improve morbidity and mortality in HFpEF are lacking. Cell-based therapies promote cardiac repair in ischemic and non-ischemic cardiomyopathies. We hypothesized that cell-based therapy ameliorates CKD-induced HFpEF. Methods and Results Yorkshire pigs (n = 26) underwent 5/6 embolization-mediated nephrectomy. CKD was confirmed by increased creatinine and decreased glomerular filtration rate (GFR). Mean arterial pressure (MAP) was not different between groups from baseline to 4 weeks. HFpEF was evident at 4 weeks by increased LV mass, relative wall thickening, end-diastolic pressure, and end-diastolic pressure-volume relationship, with no change in ejection fraction (EF). Four weeks post-embolization, allogeneic (allo) bone marrow-derived mesenchymal stem cells (MSC; 1 × 107 cells), allo-kidney-derived stem cells (KSC; 1 × 107 cells), allo-cell combination therapy (ACCT; MSC + KSC; 1:1 ratio; total = 1 × 107 cells), or placebo (Plasma-Lyte) was delivered via intra-renal artery. Eight weeks post-treatment, there was a significant increase in MAP in the placebo group (21.89 ± 6.05 mmHg) compared to the ACCT group. GFR significantly improved in the ACCT group. EF, relative wall thickness, and LV mass did not differ between groups at 12 weeks. EDPVR improved in the ACCT group, indicating decreased ventricular stiffness. Conclusions Intra-renal artery allogeneic cell therapy was safe in a CKD swine model manifesting the characteristics of HFpEF. The beneficial effect on renal function and ventricular compliance in the ACCT group supports further research of cell therapy for cardiorenal syndrome.

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

This study aims to clarify the immunogenicity in acquired and innate immune responses of cultured human corneal endothelial cells (hCECs) applied for cell injection therapy, a newly established modality for corneal endothelium failures. Thirty-four patients with corneal endothelial failure received injection of allogeneic hCEC suspension into anterior chamber. No sign of immunological rejection was observed in all 34 patients during the 5–8 years postoperative follow-up period. Cell injection therapy was successful in 2 patients treated for endothelial failure after penetrating keratoplasty and one patient with Descemet membrane stripping automated endothelial keratoplasty failure. ELISPOT assays performed in allo-mixed lymphocyte reaction to the alloantigen identical to that on the injected hCECs, elicited sparse IFN-γ-specific spots in the peripheral blood mononuclear cells of patients who received hCEC injection. The therapy generated simple and smooth graft-host junctions without wound stress. The injection of C57BL/6 CECs into the anterior chamber of BALB/c mice, which rejected C57BL/6 corneas 6 weeks ago, induced no sign of inflammatory reactions after the second challenge of alloantigen. Collectively, injection of the hCEC cell suspension in the aqueous humor induces immune tolerance that contributes to the survival of the reconstituted endothelium.

Purpose MicroRNA could be biomarker and therapeutic target for rejection. The aim of this study was to investigate the role of miR-142-5p in allogeneic immune responses using in vitro and in vivo models. Materials and Methods Primary and immortalized human umbilical vein endothelial cells (HUVECs) were cultured with unrelated blood mononuclear cells to induce allogeneic immune responses. Syngeneic and allogeneic skin graft was performed in mice. Flow cytometry, quantitative reverse transcription–polymerase chain reaction, and Western blotting was performed to understand the underlying mechanisms. Results miR-142-5p was up-regulated in primary HUVEC and a HUVEC line when allogeneic immune responses were elicited. miR-142-5p was also up-regulated in the murine allogeneic skin graft. Overexpression of miR-142-5p in HUVEC increased the expression of HLA-ABC and HLA-DR additively to allogeneic immune responses, suggesting a possible increase in alloantigen presentation. Inhibition of miR-142-5p reduced the expression of HLA-DR. ZEB1, a putative target gene of miR-142-5p, was down-regulated in HUVEC on allogeneic immune response as well as in murine allogeneic skin graft. Conclusion These results suggest that the up-regulation of miR-142-5p on allogeneic immune response might facilitate endothelial activation to exacerbate rejection.

Transfusion of red blood cells (RBCs) is a common life-saving clinical practice in severely anemic or hemorrhagic patients; however, it may result in serious pathological complications such as transfusion-related acute lung injury. The factors mediating the deleterious effects of RBC transfusion remain unclear. In this study, we tested the effects of washed long-term (RBC-O; >28 days) versus short-term (RBC-F

Intravitreal transplantation of allogeneic human retinal progenitor cells (hRPCs) holds promise as a treatment for blinding retinal degenerations. Prior work has shown that neural progenitors are well-tolerated as allografts following single injections; however, sequential delivery of allogeneic cells raises the potential risk of host sensitization with subsequent immune rejection of grafts. The current study was designed to assess whether an immune response would be induced by repeated intravitreal transplants of allogeneic RPCs utilizing the mouse animal model. We injected murine retinal progenitor cells (gmRPCs), originally derived from donors with a C57BL/6 genetic background, into BALB/c recipient mice in order to provide safety data as to what might be expected following repeated treatment of patients with allogeneic human cell product. Immune responses to gmRPCs were mild, consisting of T cells, B cells, neutrophils, and natural killer cells, with macrophages clearly the predominating. Animals treated with repeat doses of gmRPCs did not show evidence of sensitization, nor was there immune-mediated destruction of the grafts. Despite the absence of immunosuppressive treatments, allogeneic gmRPC grafts survived following repeat dosing, thus providing support for the preliminary observation that repeated injection of allogeneic RPCs to the vitreous cavity is tolerated in patients with retinitis pigmentosa.

Background: Cardiovascular surgery is confronted by a lack of suitable materials for patch repair. Acellular animal tissues serve as an abundant source of promising biomaterials. The aim of our study was to explore the bio-integration of decellularized or recellularized pericardial matrices in vivo. Methods: Porcine (allograft) and ovine (heterograft, xenograft) pericardia were decellularized using 1% sodium dodecyl sulfate ((1) Allo-decel and (2) Xeno-decel). We used two cell types for pressure-stimulated recellularization in a bioreactor: autologous adipose tissue-derived stromal cells (ASCs) isolated from subcutaneous fat of pigs ((3) Allo-ASC and (4) Xeno-ASC) and allogeneic Wharton’s jelly mesenchymal stem cells (WJCs) ((5) Allo-WJC and (6) Xeno-WJC). These six experimental patches were implanted in porcine carotid arteries for one month. For comparison, we also implanted six types of control patches, namely, arterial or venous autografts, expanded polytetrafluoroethylene (ePTFE Propaten® Gore®), polyethylene terephthalate (PET Vascutek®), chemically stabilized bovine pericardium (XenoSure®), and detoxified porcine pericardium (BioIntegral® NoReact®). The grafts were evaluated through the use of flowmetry, angiography, and histological examination. Results: All grafts were well-integrated and patent with no signs of thrombosis, stenosis, or aneurysm. A histological analysis revealed that the arterial autograft resembled a native artery. All other control and experimental patches developed neo-adventitial inflammation (NAI) and neo-intimal hyperplasia (NIH), and the endothelial lining was present. NAI and NIH were most prominent on XenoSure® and Xeno-decel and least prominent on NoReact®. In xenografts, the degree of NIH developed in the following order: Xeno-decel > Xeno-ASC > Xeno-WJC. NAI and patch resorption increased in Allo-ASC and Xeno-ASC and decreased in Allo-WJC and Xeno-WJC. Conclusions: In our setting, pre-implant seeding with ASC or WJC had a modest impact on vascular patch remodeling. However, ASC increased the neo-adventitial inflammatory reaction and patch resorption, suggesting accelerated remodeling. WJC mitigated this response, as well as neo-intimal hyperplasia on xenografts, suggesting immunomodulatory properties.

Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo. NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for “off-the-shelf” allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy.

Individuals with autism spectrum disorder (ASD) suffer from developmental disabilities that impact communication, behavior, and social interaction. Immune dysregulation and inflammation have been linked to children with ASD, the latter manifesting in serum levels of macrophage-derived chemokine (MDC) and thymus, and activation-regulated chemokine (TARC). Mesenchymal stem cells derived from umbilical cord tissue (UC-MSCs) have immune-modulatory and anti-inflammatory properties, and have been safely used to treat a variety of conditions. This study investigated the safety and efficacy of UC-MSCs administered to children diagnosed with ASD. Efficacy was evaluated with the Autism Treatment Evaluation Checklist (ATEC) and the Childhood Autism Rating Scale (CARS), and with measurements of MDC and TARC serum levels. Twenty subjects received a dose of 36 million intravenous UC-MSCs every 12 weeks (four times over a 9-month period), and were followed up at 3 and 12 months after treatment completion. Adverse events related to treatment were mild or moderate and short in duration. The CARS and ATEC scores of eight subjects decreased over the course of treatment, placing them in a lower ASD symptom category when compared with baseline. MDC and TARC inflammatory cytokine levels also decreased for five of these eight subjects. The mean MDC, TARC, ATEC, and CARS values attained their lowest levels 3 months after the last administration. UC-MSC administration in children with ASD was therefore determined to be safe. Although some signals of efficacy were observed in a small group of children, possible links between inflammation levels and ASD symptoms should be further investigated. Stem Cells Translational Medicine 2019;8:1008-1016.

Summary Cell-based immunotherapy has become the new-generation cancer medicine, and “off-the-shelf” cell products that can be manufactured at large scale and distributed readily to treat patients are necessary. Invariant natural killer T (iNKT) cells are ideal cell carriers for developing allogeneic cell therapy because they are powerful immune cells targeting cancers without graft-versus-host disease (GvHD) risk. However, healthy donor blood contains extremely low numbers of endogenous iNKT cells. Here, by combining hematopoietic stem cell (HSC) gene engineering and in vitro differentiation, we generate human allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells at high yield and purity; these cells closely resemble endogenous iNKT cells, effectively target tumor cells using multiple mechanisms, and exhibit high safety and low immunogenicity. These cells can be further engineered with chimeric antigen receptor (CAR) to enhance tumor targeting or/and gene edited to ablate surface human leukocyte antigen (HLA) molecules and further reduce immunogenicity. Collectively, these preclinical studies demonstrate the feasibility and cancer therapy potential of AlloHSC-iNKT cell products and lay a foundation for their translational and clinical development., Graphical abstract, Highlights • Allogeneic HSC-iNKT cells are generated in vitro at high yield and purity • Allogeneic HSC-iNKT cells effectively target tumor cells using multiple mechanisms • Allogeneic HSC-iNKT cells exhibit high safety and low immunogenicity • A preclinical study demonstrated feasibility, safety, and cancer therapy potential, Li et al. report the preclinical development of allogeneic-hematopoietic-stem-cell-engineered invariant natural killer T (HSC-iNKT) cells for off-the-shelf cancer therapy. Allogeneic HSC-iNKT cells demonstrate cancer therapy potential and a high safety profile.

Cell-based cancer immunotherapy has revolutionized the treatment of hematological malignancies. Specifically, autologous chimeric antigen receptor-engineered T (CAR-T) cell therapies have received approvals for treating leukemias, lymphomas, and multiple myeloma following unprecedented clinical response rates. A critical barrier to the widespread usage of current CAR-T cell products is their autologous nature, which renders these cellular products patient-selective, costly, and challenging to manufacture. Allogeneic cell products can be scalable and readily administrable but face critical concerns of graft-versus-host disease (GvHD), a life-threatening adverse event in which therapeutic cells attack host tissues, and allorejection, in which host immune cells eliminate therapeutic cells, thereby limiting their antitumor efficacy. In this review, we discuss recent advances in developing stem cell-engineered allogeneic cell therapies that aim to overcome the limitations of current autologous and allogeneic cell therapies, with a special focus on stem cell-engineered conventional αβ T cells, unconventional T (iNKT, MAIT, and γδ T) cells, and natural killer (NK) cells.

Whether perioperative allogeneic blood transfusion (PABT) negatively influences patient survival after hepatectomy (HR) for hepatocellular carcinoma (HCC) remains controversial. Five hundred two patients who underwent HR for initial HCC between 1994 and 2015 were enrolled in this study. All patients were divided into two groups: the PABT group and the non-PABT group. Differences of clinicopathological factors, overall survival (OS), recurrence-free survival (RFS), and the recurrence pattern between the two groups were evaluated. Using propensity score matching for tumor-related factors, liver functions, and surgical factors (total 11 factors), the survival impact of PABT was also analyzed. In the entire cohort, 78 patients (15.5%) received PABT such as red cell concentrate, fresh-frozen plasma, or platelets. OS (5-year OS: 55% vs. 76%; p = 0.0005) and RFS (2-year RFS: 47% vs. 56%; p = 0.0131) were significantly worse in the PABT group. The extrahepatic recurrence happened more frequently in the PABT group (15% vs. 5.4%; p = 0.0039). There were many significant clinicopathological differences between the two groups: more advanced tumor stage (tumor diameter, stage III or IV, microvascular invasion), worse liver functions (albumin, indocyanine green retention rate at 15 min), and more surgical stress (blood loss, operation time) in the PABT group. After propensity score matching, 43 pairs of patients were extracted. In this matched cohort, the survival curves of the PABT and non-PABT groups almost completely overlapped both in OS (5-year OS: 62% vs. 62%; p = 0.4384) and in RFS (2-year RFS: 49% vs. 47%; p = 0.8195). The significant difference of the extrahepatic recurrence rate disappeared in the matched cohort (p = 0.5789). Using propensity score matching, we found that PABT does not influence patient survival after HR for HCC.

Background Previous studies have identified IFNγ as an important early barrier to oncolytic viruses including vaccinia. The existing innate and adaptive immune barriers restricting oncolytic virotherapy, however, can be overcome using autologous or allogeneic mesenchymal stem cells as carrier cells with unique immunosuppressive properties. Methods To test the ability of mesenchymal stem cells to overcome innate and adaptive immune barriers and to successfully deliver oncolytic vaccinia virus to tumor cells, we performed flow cytometry and virus plaque assay analysis of ex vivo co-cultures of stem cells infected with vaccinia virus in the presence of peripheral blood mononuclear cells from healthy donors. Comparative analysis was performed to establish statistically significant correlations and to evaluate the effect of stem cells on the activity of key immune cell populations. Results Here, we demonstrate that adipose-derived stem cells (ADSCs) have the potential to eradicate resistant tumor cells through a combination of potent virus amplification and sensitization of the tumor cells to virus infection. Moreover, the ADSCs demonstrate ability to function as a virus-amplifying Trojan horse in the presence of both autologous and allogeneic human PBMCs, which can be linked to the intrinsic immunosuppressive properties of stem cells and their unique potential to overcome innate and adaptive immune barriers. The clinical application of ready-to-use ex vivo expanded allogeneic stem cell lines, however, appears significantly restricted by patient-specific allogeneic differences associated with the induction of potent anti-stem cell cytotoxic and IFNγ responses. These allogeneic responses originate from both innate (NK)- and adaptive (T)- immune cells and might compromise therapeutic efficacy through direct elimination of the stem cells or the induction of an anti-viral state, which can block the potential of the Trojan horse to amplify and deliver vaccinia virus to the tumor. Conclusions Overall, our findings and data indicate the feasibility to establish simple and informative assays that capture critically important patient-specific differences in the immune responses to the virus and stem cells, which allows for proper patient-stem cell matching and enables the effective use of off-the-shelf allogeneic cell-based delivery platforms, thus providing a more practical and commercially viable alternative to the autologous stem cell approach.

To describe the clincopathological features and evaluate risk factors of post-transplant lymphoproliferative disorder (PTLD) after allogeneic haematopoietic stem cell transplants (allo-HSCT), with comparison between paediatric and adult .Clinicopathological features of 81 cases of PTLD after allo-HSCT were analysed by histopatholgy, immunohistochemistry and in situ hybridisatioin.The cases included 58 males and 23 females with a median age of 26.7 years (range 6–55 years) and the PTLDs developed 1–60 months post-transplant (mean 5.9 months). The histological types indicated 10 cases of non-destructive PTLD, including 4 of plasmacytic hyperplasia, 5 of infectious mononucleosis and 1 of florid follicular hyperplasia. Fifty-six cases were polymorphic PTLD, and 15 were monomorphic PTLD, including thirteen of diffuse large B cell lymphoma, 1 of extranodal nasal type natural killer (NK)/T cell lymphoma and 1 of plasmablastic lymphoma. Foci and sheets of necrosis were observed in 31 cases. The infected ratio of Epstein-Barr virus (EBV) was 91.4%. Some cases were treated by reduction of immunosuppression, antiviral therapy, donor lymphocyte infusion or anti-CD20 monoclonal rituximab. Thirty-three cases died. Compared with that of adult, overall survival of paediatric recipient may be better.The first half year after allo-HSCT is very important for the development of PTLD. Type of PTLD, EBV infection and graft-versus-host disease are risk factors. The prognosis of PTLD is poor, and PTLD after allo-HSCT exhibits some features different from that after solid organ transplantation and some differences existing between adult and paediatric recipients.

Immunotherapy with chimeric antigen receptor engineered-T cells (CAR-T) has revolutionized the landscape of treatment of relapsed or refractory B-cell. However, the use of autologous T cells has limitations: variable quality of collected effector T cells, duration of the process sometimes incompatible with uncontrolled hemopathy, limited number of available CAR cells, sometimes fatal toxicities, extremely high cost. Natural Killer (NK) cells are an interesting alternative to T cells. NK cells are very powerful cytotoxic effectors that have demonstrated an anti-tumor effect after haploidentical hematopoietic stem cells transplantation or in adoptive cell therapy against a number of solid or hematological tumors. Mainly, they can be used in allogeneic situations without causing major toxic side effects. The sources of NK cells are multiple: cell line, cord blood, peripheral blood, induced pluripotent stem cells. Recent advances in manufacturing engineered CAR-NK cells make it possible to promote antibody-dependent cell-mediated cytotoxicity (ADCC), as well as the activation and persistence of these cells, notably via the cytokine Il-15. The majority of the reports on CAR-NK cells concern pre-clinical or early clinical trials. However, the many advantages of "off-the-shelf" allogeneic CAR-NK cells provide great potential in cancer treatments.

Cellular immunotherapy using chimeric antigen receptors (CARs) so far has almost exclusively used autologous peripheral blood-derived T cells as immune effector cells. However, harvesting sufficient numbers of T cells is often challenging in heavily pre-treated patients with malignancies and perturbed hematopoiesis and perturbed hematopoiesis. Also, such a CAR product will always be specific for the individual patient. In contrast, NK cell infusions can be performed in non-HLA-matched settings due to the absence of alloreactivity of these innate immune cells. Still, the infused NK cells are subject to recognition and rejection by the patient’s immune system, thereby limiting their life-span in vivo and undermining the possibility for multiple infusions. Here, we designed genome editing and advanced lentiviral transduction protocols to render primary human NK cells unsusceptible/resistant to an allogeneic response by the recipient’s CD8⁺ T cells. After knocking-out surface expression of HLA class I molecules by targeting the B2M gene via CRISPR/Cas9, we also co-expressed a single-chain HLA-E molecule, thereby preventing NK cell fratricide of B2M-knockout (KO) cells via “missing self”-induced lysis. Importantly, these genetically engineered NK cells were functionally indistinguishable from their unmodified counterparts with regard to their phenotype and their natural cytotoxicity towards different AML cell lines. In co-culture assays, B2M-KO NK cells neither induced immune responses of allogeneic T cells nor re-activated allogeneic T cells which had been expanded/primed using irradiated PBMNCs of the respective NK cell donor. Our study demonstrates the feasibility of genome editing in primary allogeneic NK cells to diminish their recognition and killing by mismatched T cells and is an important prerequisite for using non-HLA-matched primary human NK cells as readily available, “off-the-shelf” immune effectors for a variety of immunotherapy indications in human cancer. OA Förderung 2021

COVID-19 disease caused by the SARS-CoV-2 virus is characterized by dysregulation of effector T cells and accumulation of exhausted T cells. T cell responses to viruses can be corrected by adoptive cellular therapy using donor-derived virus-specific T cells. One approach is the establishment of banks of HLA-typed virus-specific T cells for rapid deployment to patients. Here we show that SARS-CoV-2–exposed blood donations contain CD4 and CD8 memory T cells which recognize SARS-CoV-2 spike, nucleocapsid and membrane antigens. Peptides of these antigens can be used to isolate virus-specific T cells in a GMP-compliant process. The isolated T cells can be rapidly expanded using GMP-compliant reagents for use as an allogeneic therapy. Memory and effector phenotypes are present in the selected virus-specific T cells, but our method rapidly expands the desirable central memory phenotype. A manufacturing yield ranging from 1010 to 1011 T cells can be obtained within 21 days culture. Thus, multiple therapeutic doses of virus-specific T cells can be rapidly generated from convalescent donors for potential treatment of COVID-19 patients.

Adoptive cellular immunotherapy using immune cells expressing chimeric antigen receptors (CARs) has shown promise, particularly for the treatment of hematological malignancies. To date, the majority of clinically evaluated CAR cell products have been derived from autologous immune cells. While this strategy can be effective it also imposes several constraints regarding logistics. This includes i) availability of center to perform leukapheresis, ii) necessity for shipment to and from processing centers, and iii) time requirements for product manufacture and clinical release testing. In addition, previous cytotoxic therapies can negatively impact the effector function of autologous immune cells, which may then affect efficacy and/or durability of resultant CAR products. The use of allogeneic CAR cell products generated using cells from healthy donors has the potential to overcome many of these limitations, including through generation of “off the shelf” products. However, allogeneic CAR cell products come with their own challenges, including potential to induce graft-versus-host-disease, as well as risk of immune-mediated rejection by the host. Here we will review promises and challenges of allogeneic CAR immunotherapies, including those being investigated in preclinical models and/or early phase clinical studies.

Chimeric antigen receptor (CAR) T-cell therapy represents a major breakthrough in the field of hematology. "Off-the-shelf" allogeneic CAR T-cells from donors have many potential advantages over autologous approaches, such as the immediate availability of cryopreserved batches, possible standardization of the cell product, time for multiple cell modifications, redosing and decreased cost. However, allogeneic T-cells possess foreign immunological identities that can lead to graft-versus-host disease (GvHD) and their rejection by the host immune system. In this review, we describe the different approaches to produce allogeneic CAR T-cells with limited potential for GvHD and that can persist in the recipient. The preliminary clinical results obtained with the first generation of allogeneic CAR T-cells are presented as well as the perspectives in hematological malignancies and solid tumors.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes cells that recognize glycolipid antigens associated with CD1d, non-classical antigen presenting proteins. They can drive either pro-inflammatory (Th-1) or anti-inflammatory (Th-2) immune microenvironment through the production of both Th-1 and Th-2 type cytokines upon activation, thus play a vital role in cancer, infection, and autoimmune diseases. Adoptive cell therapy using ex vivo expanded iNKT cells is a promising approach to enhance anti-tumor immunity or immunosuppression. However, overcoming phenotypic and functional heterogeneity and promoting in vivo persistency of iNKT cells remains to be a challenge. Here, we compared various methods for ex vivo expansion of human iNKT cells and assessed the quality of expansion, phenotype, and cytokine production profile of expanded iNKT cells. While a direct stimulation of iNKT cells in peripheral blood mononuclear cells with agonist glycolipid led to the expansion of iNKT cells in varying degrees, stimulation of enriched iNKT cells by irradiated autologous peripheral blood mononuclear cells or allogeneic dendritic cells resulted in consistent expansion of highly pure iNKT cells. Interestingly, the mode of antigenic stimulation influenced the dominant subtype of expanded iNKT cells. Further, we evaluated whether additional IL-7 or IL-15 during antigenic stimulation with allogeneic dendritic cells can improve the phenotypic heterogeneity and modify cytokine production profile of iNKT cells expanded from 18 consecutive donors. The presence of IL-7 or IL-15 during antigenic stimulation did not affect the fold of expansion or purity of expanded iNKT cells. However, IL-7, but not IL-15, led to a better expansion of CD4+ iNKT cells, enhanced Th-2 type cytokine production of CD4+ iNKT cells, and maintained the expansion of central memory (CD45RA-CD62L+) CD4+ iNKT cells. Our results suggest the addition of IL-7 during antigenic stimulation with allogeneic dendritic cells can promote the expansion of CD62L+Th-2+CD4+ human iNKT cells that can be used as novel immunotherapeutic to control excessive inflammation to treat various autoimmune diseases.

Immunotherapy has limited efficacy against locally advanced pancreatic cancer (LAPC) due to the presence of an immunosuppressive microenvironment (ISM). Irreversible electroporation (IRE) can not only induce immunogenic cell death, but also alleviate immunosuppression. This study aimed to investigate the antitumor efficacy of IRE plus allogeneic γδ T cells in LAPC patients. A total of 62 patients who met the eligibility criteria were enrolled in this trial, then randomized into two groups (A: n = 30 and B: n = 32). All patients received IRE therapy and after receiving IRE, the group A patients received at least two cycles of γδ T-cell infusion as one course continuously. Group A patients had better survival than group B patients (median OS: 14.5 months vs. 11 months; median PFS: 11 months vs. 8.5 months). Moreover, the group A patients treated with multiple courses of γδ T-cell infusion had longer OS (17 months) than those who received a single course (13.5 months). IRE combined with allogeneic γδ T-cell infusion is a promising strategy to enhance the antitumor efficacy in LAPC patients, yielding extended survival benefits.ClinicalTrials.gov ID: NCT03180437.

Tumor cell membrane-derived nanostructures targeting homologous tumors are promising biomimetic drugs. Herein, curcumin (Cur) and chlorin e6 (Ce6) were co-loaded into PLGA nanoparticles (NPs), and then the NPs were coated with MCF-7 cell membranes (MCNPs). Cell membrane coating sharply increased the uptake of MCNPs by homologous cells, as compared to that with naked NPs. The NPs co-loaded with Cur and Ce6 (Cur/Ce6-NPs) showed a stronger proliferation-inhibitory effect on MCF-7 cells than the NP groups loaded with Cur and Ce6 alone. Cytotoxicity and apoptosis rates of MCF-7 cells in the Cur/Ce6-MCNPs group were significantly higher than those in the uncoated Cur/Ce6-NPs group. Both Cur/Ce6-NPs and Cur/Ce6-MCNPs significantly inhibited the migration of MCF-7cells, although Cur/Ce6-MCNPs showed a stronger effect. Compared to that of Cur/Ce6-NPs, the elimination of Cur/Ce6-MCNPs was both decreased and retarded, prolonging their in vivo systemic circulation and resulting in improved bioavailability. After intravenous administration for 24 h, the fluorescence intensity of drugs in the liver and spleen of the Cur/Ce6-MCNPs group was significantly weaker than that in the Cur/Ce6-NPs group, but that in tumor tissue was enhanced. Further, Cur/Ce6-MCNPs treatment achieved significantly better tumor-suppressive effects in vivo than Cur/Ce6-NPs, resulting in smaller tumor weights, increased apoptosis rates, and the down regulation of Ki67 protein in the tumor tissue. Thus, the tumor cell membrane-camouflaged nanocomposites have potential for homologous tumor-targeted therapy. Furthermore, photodynamic therapy combined with chemotherapy has promising future prospects.

The CSF-470 vaccine consists of lethally-irradiated allogeneic cells derived from four cutaneous melanoma cell lines administered plus BCG and GM-CSF as adjuvants. In an adjuvant phase II study vs. IFN-α2b, the vaccine significantly prolonged the distant metastasis-free survival (DMFS) of stages IIB-IIC-III melanoma patients with evidence of the induction of immune responses against vaccine cells. Purpose: The aim of this study was to analyze the antigens against which the immune response was induced, as well as the T-helper profile and lytic ability of immune cells after CSF-470 treatment. Methods: HLA-restricted peptides from tumor-associated antigens (TAAs) were selected from TANTIGEN database for 13 evaluable vaccinated patients. In addition, for patient #006 (pt#006), tumor somatic variants were identified by NGS and candidate neoAgs were selected by predicted HLA binding affinity and similarity between wild type (wt) and mutant peptides. The patient‘s PBMC reactivity against selected peptides was detected by IFNγ-ELISPOT. T-helper transcriptional profile was determined by quantifying GATA-3, T-bet, and FOXP3 mRNA by RT-PCR, and intracellular cytokines were analyzed by flow cytometry. Autologous tumor cell lysis by PBMC was assessed in an in vitro calcein release assay. Results: Vaccinated patient‘s PBMC reactivity against selected TAAs derived peptides showed a progressive increase in the number of IFNγ-producing cells throughout the 2-yr vaccination protocol. ELISPOT response correlated with delayed type hypersensitivity (DTH) reaction to CSF-470 vaccine cells. Early upregulation of GATA-3 and Foxp3 mRNA, as well as an increase in CD4+IL4+cells, was associated with a low DMFS. Also, IFNγ response against 9/73 predicted neoAgs was evidenced in the case of pt#006; 7/9 emerged after vaccination. We verified in pt# 006 that post-vaccination PBMC boosted in vitro with the vaccine lysate were able to lyse autologous tumor cells. Conclusions: A progressive increase in the immune response against TAAs expressed in the vaccine and in the patient's tumor was induced by CSF-470 vaccination. In pt#006, we demonstrated immune recognition of patient's specific neoAgs, which emerged after vaccination. These results suggest that an initial response against shared TAAs could further stimulate an immune response against autologous tumor neoAgs. Fil: Podaza, Enrique Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina Fil: Carri, Ibel. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Aris, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina Fil: Von Euw, Erika María. University of California at Los Angeles; Estados Unidos. T-Cure Bioscience; Argentina Fil: Bravo, Alicia Ines. Gobierno de la Provincia de Buenos Aires. Hospital El Cruce Doctor Néstor Carlos Kirchner. Centro de Medicina Traslacional; Argentina Fil: Blanco, Paula. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina Fil: Ortiz Wilczyñski, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Koile, Daniel Isaac. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Yankilevich, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Mordoh, Jose. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Instituto Alexander Fleming.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Barrio, Maria Marcela. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

EgeUn###, Objectives: The only known curative therapy for primary myelofibrosis is allogeneic hematopoietic stem cell transplant. Materials and Methods: We retrospectively evaluated 11 transplant procedures involving 10 patients (5 men and 5 women) diagnosed with primary myelofibrosis between 2005 and 2014. Results: The median age at the time of transplant was 60.5 years (range, 22-62 years). Stem cell sources were unrelated (n = 1) and related (n = 11) peripheral blood stem cells. Conditioning regimen was myeloablative for 8 and reduced intensity for 3 transplants. The median number of infused CD34+ cells was 6.8 x 10(6) cells/kg (range, 3.2-10.4 x 10(6) cells/kg). Neutrophil and platelet engraftment occurred at median of 22 days (range, 12-31 days) and 19.5 days (range, 13-56 days). Acute and chronic graft-versus-host disease was seen in 4 of 11 allografts. Relapse and nonrelapse mortality rates were 20%. Six patients (60%) were still alive without disease after median follow-up of 68.5 months (range, 17-120 months). Median progression-free survival and overall survival were 61 months (range, 2-120 months) and 65 months (range, 2-120 months). Conclusions: Our results suggest that allogeneic hematopoietic stem cell transplant may provide a curative treatment for primary myelofibrosis patients. A myeloablative regimen seems to be effective and safe, especially for younger primary myelofibrosis patients.

ObjectiveTo assess the feasibility, safety, and efficacy of intracoronary allogeneic cardiosphere-derived cells (CAP-1002) in patients with Duchenne muscular dystrophy (DMD).MethodsThe Halt Cardiomyopathy Progression (HOPE)-Duchenne trial is a phase I/II, randomized, controlled, open-label trial (NCT02485938). Patients with DMD >12 years old, with substantial myocardial fibrosis, were randomized (1:1) to usual care (control) or global intracoronary infusion of CAP-1002 (75 million cells). Participants were enrolled at 3 US medical centers between January and August 2016 and followed for 12 months. An independent Data and Safety Monitoring Board provided safety oversight. Cardiac function and structure were assessed by MRI, and analyzed by a blinded core laboratory. Skeletal muscle function was assessed by performance of the upper limb (PUL).ResultsTwenty-five eligible patients (mean age 17.8 years; 68% wheelchair-dependent) were randomized to CAP-1002 (n = 13) or control (n = 12). Incidence of treatment-emergent adverse events was similar between groups. Compared to baseline, MRI at 12 months revealed significant scar size reduction and improvement in inferior wall systolic thickening in CAP-1002 but not control patients. Mid-distal PUL improved at 12 months in 8 of 9 lower functioning CAP-1002 patients, and no controls (p = 0.007).ConclusionsIntracoronary CAP-1002 in DMD appears safe and demonstrates signals of efficacy on both cardiac and upper limb function for up to 12 months. Thus, future clinical research on CAP-1002 treatment of DMD cardiac and skeletal myopathies is warranted.Classification of evidenceThis phase I/II study provides Class II evidence that for patients with DMD, intracoronary CAP-1002 is feasible and appears safe and potentially effective.

Regulatory T cells play an important role in the control of autoimmune diseases and maintenance of tolerance. In the context of transplantation, regulatory T cells (Tregs) have been proposed as new therapeutic tools that may induce allospecific tolerance toward the graft, avoiding the side effects induced by generalized immunosuppressors. Although most clinical trials are based on the use of thymic Tregs in adoptive therapy, some reports suggest the potential use of in vitro induced Tregs (iTregs), based on their functional stability under inflammatory conditions, indicating an advantage in a setting of allograft rejection. The aim of this work was to generate and expand large numbers of allospecific Tregs that maintain stable suppressive function in the presence of pro-inflammatory cytokines. Dendritic cells were derived from monocytes isolated from healthy donors and were co-cultured with CTV-labeled naïve T cells from unrelated individuals, in the presence of TGF-β1, IL-2, and retinoic acid. After 7 days of co-culture, proliferating CD4+CD25++CTV− cells (allospecific iTregs) were sorted and polyclonally expanded for 6 weeks in the presence of TGF-β1, IL-2, and rapamycin. After 6 weeks of polyclonal activation, iTregs were expanded 230,000 times, giving rise to 4,600 million allospecific iTregs. Allospecific iTregs were able to specifically suppress the proliferation of autologous CD4+ and CD8+ T cells in response to the allo-MoDCs used for iTreg generation, but not to third-party allo-MoDCs. Importantly, 88.5% of the expanded cells were CD4+CD25+FOXP3+, expressed high levels of CCR4 and CXCR3, and maintained their phenotype and suppressive function in the presence of TNF-α and IL-6. Finally, analysis of the methylation status of the FOXP3 TSDR locus demonstrated a 40% demethylation in the purified allospecific iTreg, prior to the polyclonal expansion. Interestingly, the phenotype and suppressive activity of expanded allospecific iTregs were maintained after 6 weeks of expansion, despite an increase in the methylation status of the FOXP3 TSDR. In conclusion, this is the first report that demonstrates a large-scale generation of allospecific iTregs that preserve a stable phenotype and suppressor function in the presence of pro-inflammatory cytokines and pave the way for adoptive cell therapy with iTregs in transplanted patients.

Introduction: Progress in allogeneic hematopoietic stem cell transplantation (HSCT) procedures has been associated with improved survival in HSCT recipients. However, they have also brought to light organ-specific complications, especially pulmonary complications. In this setting, pulmonary complications are consistently associated with poor outcomes, and improved management of these complications is required. Areas covered: We review the multiple infectious and noninfectious lung complications that occur both early and late after allogeneic HSCT. This includes the description of these complications, risk factors, diagnostic approach and outcome. A literature search was performed using PubMed-indexed journals. Expert commentary: Multiple lung complications after allogeneic HSCT can be diagnosed concomitantly and require a multidisciplinary approach. A specific clinical evaluation including a precise analysis of a lung CT scan is necessary. Management of these lung complications, especially the noninfectious ones, is impaired by the lack of prospective, randomized control trials, suggesting preventive strategies should be developed.

BACKGROUND AND OBJECTIVE Extracorporeal photopheresis (ECP) is an important immune tolerance inducing therapy for graft-versus-host disease (GvHD). However, a sufficient number of ECP cycles cannot be performed in patients with severe GvHD and contraindications for apheresis. Allogeneic sources of leucocytes for use as ECP treatment would be of great benefit. Therefore, this study aimed to test the therapeutic potential of novel sources of leucocytes for ECP. MATERIALS AND METHODS Graft-versus-host disease mice were treated with ECP using therapeutic cells from different allogeneic sources. Splenocytes were incubated with 8-methoxypsoralen (8-MOP), irradiated with UVA light and injected into GvHD mice as a model for ECP. RESULTS The therapy with 8-MOP/UVA-treated cells from healthy mice of the bone marrow transplantation (BMT) donor strain reduced the GvHD symptoms, at least in a model of chronic GvHD. In the acute GvHD model, 8-MOP/UVA-treated cells from the BMT donor or recipient strain did not show significant improvements in GvHD symptoms or survival time. Pre-activation of cells by mixed lymphocyte reactions before 8-MOP/UVA treatment also failed to result in significant differences in survival time or GvHD score. In contrast, ECP with third-party 8-MOP/UVA-treated cells from a HLA-mismatched donor resulted in a mean survival time of 37 days compared to 21 days in the control group. CONCLUSION In our analysis of novel allogeneic leucocyte sources for ECP, we could demonstrate that the source of the 8-MOP/UVA-treated cells is crucial. The underlying immunologic effect of allogeneic 8-MOP/UVA-treated cells needs to be investigated in future studies.

Background: The mouse brain microvascular endothelial cell line bEnd.3 was used to develop a vaccine and its anti-tumor effect on lung metastases was observed in immunized mice. Methods: Mouse bEnd.3 cells cultured in-vitro and then fixed with glutaraldehyde was used to immunize mice; mice were challenged with the metastatic cancer cell line U14, and changes in metastatic cancer tissues were observed through hematoxylin and eosin staining. Carboxyfluorescein succinimidyl amino ester (CSFE) and propidium iodide (PI) were used to detect cytotoxic activity of spleen T lymphocytes; the ratio of CD3+ and CD8+ T-cell sub-sets was determined by flow cytometry. Enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and immunoblot were used to examine the specific response of the antisera of immunized mice. Results: The number of metastatic nodules in bEnd.3 and human umbilical vein endothelial cell (HUVEC) vaccine groups was less than NIH3T3 vaccine group and phosphate buffered saline (PBS) control group. The bEnd.3-induced and HUVEC-induced cytotoxic T-lymphocytes (CTLs) showed significant lytic activity against bEnd.3 and HUVEC target cells, while the antisera of mice in bEnd.3 and HUVEC vaccine groups showed specific immune responses to membrane proteins and inhibited target cell proliferation in-vitro. Immunoblot results showed specific bands at 180KD and 220KD in bEnd.3 and at 130 kD and 220 kD in HUVEC lysates. Conclusions: Allogeneic bEnd.3 vaccine induced an active and specific immune response to tumor vascular endothelial cells that resulted in production of antibodies against the proliferation antigens VEGF-R II, integrin, Endog etc. Immunization with this vaccine inhibited lung metastasis of cervical cancer U14 cells and prolonged the survival of these mice.

Purpose: Mesothelioma has been regarded as a nonimmunogenic tumor, which is also shown by the low response rates to treatments targeting the PD-1/PD-L1 axis. Previously, we demonstrated that autologous tumor lysate–pulsed dendritic cell (DC) immunotherapy increased T-cell response toward malignant mesothelioma. However, the use of autologous tumor material hampers implementation in large clinical trials, which might be overcome by using allogeneic tumor cell lines as tumor antigen source. The purpose of this study was to investigate whether allogeneic lysate–pulsed DC immunotherapy is effective in mice and safe in humans. Experimental Design: First, in two murine mesothelioma models, mice were treated with autologous DCs pulsed with either autologous or allogeneic tumor lysate or injected with PBS (negative control). Survival and tumor-directed T-cell responses of these mice were monitored. Results were taken forward in a first-in-human clinical trial, in which 9 patients were treated with 10, 25, or 50 million DCs per vaccination. DC vaccination consisted of autologous monocyte–derived DCs pulsed with tumor lysate from five mesothelioma cell lines. Results: In mice, allogeneic lysate–pulsed DC immunotherapy induced tumor-specific T cells and led to an increased survival, to a similar extent as DC immunotherapy with autologous tumor lysate. In the first-in-human clinical trial, no dose-limiting toxicities were established and radiographic responses were observed. Median PFS was 8.8 months [95% confidence interval (CI), 4.1–20.3] and median OS not reached (median follow-up = 22.8 months). Conclusions: DC immunotherapy with allogeneic tumor lysate is effective in mice and safe and feasible in humans. Clin Cancer Res; 24(4); 766–76. ©2017 AACR.

Refined graft-versus-host disease (GVHD)/relapse-free survival (GRFS) considers main outcomes of allogeneic stem cell transplant (HSCT), estimating long-term survival without significant morbidity as a surrogate of HSCT success. We compared GRFS in 5059 adults with acute myeloid leukemia (AML), undergoing HSCT in first complete remission from 2000 to 2015 either from a matched sibling (MSD, n = 3731) or unrelated donor (MUD, n = 1328). Median age was 49 (range: 18–76) years. Median follow-up was 32 and 60 months in MSD and MUD, respectively (p < 0.01). Compared to MSD, at 4 years, MUD recipients had lower GRFS, with higher NRM, grade III–IV acute GVHD, and extensive chronic GVHD (HR: 1.42, p < 0.01). We also performed a risk factor analyses, showing unfavorable cytogenetics (HR: 1.42, p < 0.01) and peripheral blood as stem cell source (HR: 1.22, p < 0.01) associated to lower GRFS, while this was higher with in vivo T-cell depletion (TCD, HR: 0.73, p < 0.01) and shorter time from diagnosis to HSCT (HR 0.96, p < 0.01). Different factors, modifiable or not, such as donor type, stem cell source, disease biology, and in vivo TCD, impact on GRFS and this may guide in the future transplant choices to improve morbidity and long-term quality of life.

DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell–cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.

The optimal rabbit anti-thymocyte globulin (rATG) graft-versus-host disease (GVHD) prophylaxis regimen in matched sibling donor peripheral blood stem cell transplantation (MSD-PBSCT) remains to be elucidated. In this prospective study, we used low-dose rATG for GVHD prophylaxis in patients or donors aged ≥ 40 years with hematological malignancies receiving MSD-PBSCT. rATG was administered to 40 patients at an intravenous dose of 5 mg/kg divided over day 5 and day 4 before graft infusion. No graft failure occurred. Median times to leukocyte engraftment and platelet engraftment were 11.0 days and 13.9 days. The cumulative incidence of grades 2–4 and grades 3–4 acute GVHD at day +100 was 30.0% and 2.6%. The 2-year cumulative incidence of extensive chronic GVHD and severe chronic GVHD was 11.4% and 14.7%. 93.5% (29/31) of patients had discontinued immunosuppressive medication within 3 years after transplantation. The 2-year cumulative incidence of transplant-related mortality (TRM) and relapse was 14.0% and 22.6%. The cumulative incidence of cytomegalovirus reactivation, Epstein–Barr virus reactivation, and fungal infection was 22.3%, 12.9%, and 12.5%. Kaplan–Meier estimates for overall survival, disease-free survival, and GVHD-free and relapse-free survival 3 years after transplantation were 68.9%, 68.9%, and 54.0%. rATG for GVHD prophylaxis is tolerable and efficacious at a 5 mg/kg total dose administered over 2 days (days −5 to −4) in patients receiving allogeneic MSD-PBSCT.

Significance HLA genes confer the strongest genetic autoimmune disease risk. Specific HLA-DRB1 alleles predispose to rheumatoid arthritis (RA), while others encoding the sequence DERAA are RA-protective. Unexpectedly, having given birth to children with DERAA + HLA-DRB1 alleles prior to onset was found to increase RA risk in women. We show that microchimerism (Mc; allogeneic cell long-term legacy of pregnancy) carrying DERAA + HLA increases the odds of RA 17-fold and that allogeneic cells carrying DERAA stimulated DERAA −/− T cells in vitro. Microbial-derived DERAA peptides (among others) are known to stimulate DERAA-directed T cells naturally present in DERAA −/− individuals. Our data indicate that Mc with DERAA alloantigens can potentially activate similar adaptive immunity pathways and mechanistically link naturally occurring Mc to an autoimmune disorder.

Low haemocompatibility of left ventricular assist devices (LVAD) surfaces necessitates anticoagulative therapy. Endothelial cell (EC) seeding can support haemocompatibility, however, the availability of autologous ECs is limited. In contrast, allogeneic ECs are readily available in sufficient quantity, but HLA disparities induce harmful immune responses causing EC loss. In this study, we investigated the feasibility of using allogeneic low immunogenic ECs to endothelialize LVAD sintered inflow cannulas (SIC). To reduce the immunogenicity of ECs, we applied an inducible lentiviral vector to deliver short-hairpins RNA to silence HLA class I expression. HLA class I expression on ECs was conditionally silenced by up to 70%. Sufficient and comparable endothelialization rates were achieved with HLA-expressing or HLA-silenced ECs. Cell proliferation was not impaired by cell-to-Sintered Inflow Cannulas (SIC) contact or by silencing HLA expression. The levels of endothelial phenotypic and thrombogenic markers or cytokine secretion profiles remained unaffected. HLA-silenced ECs-coated SIC exhibited reduced thrombogenicity. In contrast to native ECs, HLA-silenced ECs showed lower cell lysis rates when exposed to allogeneic T cells or specific anti-HLA antibodies. Allogeneic HLA-silenced ECs could potentially become a valuable source for LVAD endothelialization to reduce immunogenicity and correspondingly the need for anticoagulative therapy which can entail severe side effects.

Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, we evaluated their inductive and modulatory capacity regarding immune responses and underlying key mechanisms in vitro. For this, cryopreserved hAACs were either cultured in the presence of interferon-gamma (IFNγ) or left unstimulated. The expression of characteristic mesenchymal stromal cell markers (CD29, CD44, CD73, CD105, CD166) was revealed by flow cytometry that also highlighted a predominant negativity for CD90. A low immunogeneic phenotype in an inflammatory milieu was shown by lacking expression of co-stimulatory molecules and upregulation of the inhibitory ligands PD-L1 and PD-L2, despite de novo expression of HLA-DR. Co-cultures of hAACs with allogeneic peripheral blood mononuclear cells, proved their low immunogeneic state by absence of induced T cell proliferation and activation. Additionally, elevated levels of IL-1β, IL-33, and IL-10 were detectable in those cell culture supernatants. Furthermore, the immunomodulatory potential of hAACs was assessed in co-cultures with αCD3/αCD28-activated peripheral blood mononuclear cells. Here, a strong inhibition of T cell proliferation and reduction of pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-17A, IL-2) were observable after pre-stimulation of hAACs with IFNγ. Transwell experiments confirmed that mostly soluble factors are responsible for these suppressive effects. We were able to identify indolamin-2,3-dioxygenase (IDO) as a potential key player through a genome-wide gene expression analysis and could demonstrate its involvement in the observed immunological responses. While the application of blocking antibodies against both PD-1 ligands did not affect the immunomodulation by hAACs, 1-methyl-L-tryptophan as specific inhibitor of IDO was able to restore proliferation and to lower apoptosis of T cells. In conclusion, hAACs represent a cardiac-derived mesenchymal stromal-like cell type with a high potential for the application in an allogeneic setting, since they do not trigger T cell responses and even increase their immunomodulatory potential in inflammatory environments.

To systematically define molecular features in human tumor cells that determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly annotated ‘DNA-barcoded’ solid tumor cell lines in multiplexed format and applied genome-scale CRISPR-based gene-editing screens in several solid tumor cell lines, to functionally interrogate which genes in tumor cells regulate the response to NK cells. In these orthogonal studies, NK cell–sensitive tumor cells tend to exhibit ‘mesenchymal-like’ transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); and low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell–sensitive tumor cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. The use of natural killer (NK) cells in immunotherapy as an alternative to allogeneic T cells is gaining ground. Here, two genome-scale high-throughput platforms are used to identify genes that modulate the sensitivity of multiple solid tumor cell lines to NK-mediated killing.

The clinical application of induced pluripotent stem cells (iPSC) needs to balance the use of an autologous source that would be a perfect match for the patient against any safety or efficacy issues that might arise with using cells from an older patient or donor. Drs. Takahashi and Yamanaka and the Office of Cellular and Tissue-based Products (PMDA), Japan, have had concerns over the existence of accumulated DNA mutations in the cells of older donors and the possibility of long-term negative effects. To mitigate the risk, they have chosen to partner with the Umbilical Cord (UC) banks in Japan to source allogeneic-matched donor cells. Production of iPSCs from UC blood cells (UCB) has been successful, however, reprogramming blood cells requires cell enrichment with columns or flow cytometry and specialized growth media. These requirements add to the cost of production and increase the manipulation of the cells, which complicates the regulatory approval process. Alternatively, umbilical cord tissue mesenchymal stromal cells (CT-MSCs) have the same advantage as UCB cells of being a source of young donor cells. Crucially, CT-MSCs are easier and less expensive to harvest and grow compared to UCB cells. Here, we demonstrate that CT-MSCs can be easily isolated without expensive enzymatic treatment or columns and reprogramed well using episomal vectors, which allow for the removal of the reprogramming factors after a few passages. Together the data indicates that CT-MSCs are a viable source of donor cells for the production of clinical-grade, patient matched iPSCs.

Supplemental Digital Content is available in the text, Introduction: Recurrent ovarian carcinoma has dismal prognosis, but control of disease and prolonged survival are possible in some patients. The estimated 5-year survival is 46% for all stages of ovarian cancer, and only 28% for metastasized disease. Notably, the majority of women with ovarian cancer are diagnosed with stage III or IV disease with a high recurrence rate. As most women with relapsed or metastatic cancer will die of progressive disease, there is an urgent need for novel therapeutic strategies. The primary aim of our study is to evaluate safety and toxicity of intraperitoneal infusion of ex vivo-expanded natural killer cells (NK), generated from CD34+ umbilical cord blood (UCB) progenitor cells, with and without a preceding non-myeloablative immunosuppressive conditioning regimen in patients suffering from recurrent ovarian cancer. The secondary objectives are to compare the in vivo lifespan, expansion, and biological activity of intraperitoneally infused NK cell products with or without preparative chemotherapy, as well as evaluate effects on disease load. Methods: In this phase I safety trial, 12 patients who are suffering from recurrent ovarian cancer, detected by a significant rise in serum level of CA-125 on two successive time points, will be included. Prior to UCB-NK cell infusion, a laparoscopy is performed to place a catheter in the peritoneal cavity. The first cohort of three patients will receive a single intraperitoneal infusion of 1.5-3×109 UCB-NK cells, generated ex vivo from CD34+ hematopoietic progenitor cells obtained from an allogeneic UCB unit, without a preparative chemotherapy regimen. The second group of three patients will be treated with a similar dose of UCB-NK cells following a preparative four days non-myeloablative immunosuppressive conditioning regimen with cyclophosphamide and fludarabine (Cy/Flu). If no severe toxicity is seen in these 6 patients, an extension cohort of 6 patients will be included to answer the secondary objectives. Discussion: This study investigates the safety of a promising new cellular therapy in a group of patients with a poor prognosis. Demonstration of safety and in vivo expansion capacity of allogeneic UCB-NK cells in the absence of Cy/Flu pretreatment will provide rationale for UCB-NK cell infusion after regular second-line chemotherapy.

The early response to bacterial infection requires cytokine responses by immune cells. In this issue of Cell Host & Microbe, Seo et al. (2018) demonstrate that TNF-TNFR superfamily molecules LIGHT and HVEM stimulate early IFN-γ production by type 3 innate lymphoid cells, which are critical for defense against Yersinia enterocolitica.

The objective of this study was to translate reaction conditions and quality control methods used for production of an astatine-211(211At)-labeled anti-CD45 monoclonal antibody (MAb) conjugate, 211At-BC8-B10, from the laboratory setting to cGMP production. Five separate materials were produced in the preparation of 211At-BC8-B10: (1) p-isothiocyanato-phenethyl-closo-decaborate(2-) (B10-NCS), (2) anti-CD45 MAb, BC8, (3) BC8-B10 MAb conjugate, (4) [211At]NaAt, and (5) 211At-BC8-B10. The 211At-labeling reagent, B10-NCS, was synthesized as previously reported. BC8 was produced, then conjugated with B10-NCS under cGMP conditions to form BC8-B10. [211At]NaAt was produced by α-irradiation of Bi targets, followed by isolation of the 211At using a "wet chemistry" method. The clinical product, 211At-BC8-B10, was prepared by reacting [211At]NaAt with BC8-B10 in NH4OAc buffer (pH 5.5) for 2 min at room temperature, followed by size-exclusion chromatography purification. Quality control tests conducted on the 211At-BC8-B10 included evaluations for purity and identity, as well as pyrogen and sterility tests. Stability of the 211At-BC8-B10 in 25 mg/mL sodium ascorbate solution was evaluated at 1, 2, 4, 6 and 21 h post isolation. For qualification, three consecutive 211At-BC8-B10 clinical preparations were successfully conducted in the cGMP suite, and an additional cGMP clinical preparation was carried out to validate each step required to deliver 211At-BC8-B10 to a patient. These cGMP preparations provided 0.80-1.28 Gbq (21.5-34.5 mCi) of 211At-BC8-B10 with radiochemical purity of >97%. The preparations were found to be sterile and have a pyrogen level 95% for up to 21 h at room temperature. The experiments conducted have defined conditions for translation of 211At-BC8-B10 production from the laboratory to cGMP suite. This study has allowed the initiation of a phase I/II clinical trial using 211At-BC8-B10 (NCT03128034).

Background: Each year, over 5 million red blood cell (RBC) transfusions are administered to patients in the USA. Despite the therapeutic benefits of RBC transfusions, there are associated risks. RBC-specific alloantibodies may form in response to antigenic differences between RBC donors and recipients; these alloantibodies can be a problem as they may mediate hemolysis or pose barriers to future transfusion support. While there is currently no reliable way to predict which RBC recipients will make an alloantibody response, risk factors such as inflammation have been shown to correlate with increased rates of RBC alloimmunization. The underlying mechanisms behind how inflammation mediates alloantibody production are incompletely defined. Methods: To assess erythrophagocytosis, mice were treated with PBS or inflammatory stimuli followed by a transfusion of allogeneic RBCs labeled with a lipophilic dye. At multiple time points, RBC consumption and expression of activation makers by leukocytes was evaluated. To determine which antigen presenting cell (APC) subset(s) were capable of promoting allogeneic T cell activation, sorted leukocyte populations (which had participated in erythrophagocytosis) were co-cultured in vitro with allogeneic CD4+ T cells; T cell proliferation and ability to form immunological synapses with APCs were determined. Results: Upon transfusion of fresh allogeneic RBCs, multiple APCs consumed transfused RBCs. However, only CD8+ and CD11b+ dendritic cells formed productive immunological synapses with allogeneic T cells and stimulated proliferation. Importantly, allogeneic T cell activation and RBC alloantibody production occurred in response to RBC transfusion alone, and transfusion in the context of inflammation enhanced RBC consumption, the number of immune synapses, allogeneic T cell proliferation, and the rate and magnitude of alloantibody production. Conclusions: These data demonstrate that regardless of the ability to participate in RBC consumption, only a subset of APCs are capable of forming an immune synapse with T cells thereby initiating an alloantibody response. Additionally, these data provide mechanistic insight into RBC alloantibody generation.

To characterize the outcomes of patients who developed a particular subsequent solid cancer after hematopoietic cell transplantation (HCT), age at cancer diagnosis, survival, and causes of death were compared with the respective primary cancer in the general population, using data from the national HCT registry and population-based cancer registries in Japan. Among 31 867 patients who underwent a first HCT between 1990 and 2013 and had progression-free survival at 1 year, 713 patients developed subsequent solid cancer. The median age at subsequent solid cancer diagnosis was 55 years, which was significantly younger than the 67 years for primary cancer patients in the general population (P < .001). The overall survival probability was 60% at 3 years after diagnosis of subsequent solid cancer and differed according to cancer type. Development of most solid cancers was associated with an increased risk of subsequent mortality after HCT. Subsequent solid cancers accounted for 76% of causes of death. Overall survival probabilities adjusted for age, sex, and year of diagnosis were lower in the HCT population than in the general population for colon, bone/soft tissue, and central nervous system cancers and did not differ statistically for other cancers. In conclusion, most subsequent solid cancers occurred at younger ages than primary cancers, emphasizing the need for cancer screening at younger ages. Subsequent solid cancers showed similar or worse survival compared with primary cancers. Biological and genetic differences between primary and subsequent solid cancers remain to be determined.