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Background To accelerate the translation of novel immunotherapeutic treatment approaches, the development of analytic methods to assess their efficacy at early in vitro stages is necessary. Using a droplet-based microfluidic platform, we have established a method for multiparameter quantifiable phenotypic and genomic observations of immunotherapies. Chimeric antigen receptor (CAR) natural killer (NK) cells are of increased interest in the current immunotherapy landscape and thus provide an optimal model for evaluating our novel methodology.Methods For this approach, NK cells transduced with a CD19 CAR were compared with non-transduced NK cells in their ability to kill a lymphoma cell line. Using our microfluidic platform, we were able to quantify the increase in cytotoxicity and synaptic contact formation of CAR NK cells over non-transduced NK cells. We then optimized our droplet sorter and successfully used it to separate NK cells based on target cell killing to perform transcriptomic analyses.Results Our data revealed expected improvement in cytotoxicity with the CD19 CAR but more importantly, provided unique insights into the factors involved in the cytotoxic mechanisms of CAR NK cells. This demonstrates a novel, improved system for accelerating the pre-clinical screening of future immunotherapy treatments.Conclusions This study provides a new potential approach for enhanced early screening of immunotherapies to improve their development, with a highly relevant cell model to demonstrate. Additionally, our validation studies provided some potential insights into transcriptomic determinants influencing CAR NK cytotoxicity.

Natural killer (NK) cells have gained attention as a promising adoptive cell therapy platform for their potential to improve cancer treatments. NK cells offer distinct advantages over T-cells, including major histocompatibility complex class I (MHC-I)-independent tumor recognition and low risk of toxicity, even in an allogeneic setting. Despite this tremendous potential, challenges persist, such as limited in vivo persistence, reduced tumor infiltration, and low absolute NK cell numbers. This review outlines several strategies aiming to overcome these challenges. The developed strategies include optimizing NK cell expansion methods and improving NK cell antitumor responses by cytokine stimulation and genetic manipulations. Using K562 cells expressing membrane IL-15 or IL-21 with or without additional activating ligands like 4-1BBL allows “massive” NK cell expansion and makes multiple cell dosing and “off-the-shelf” efforts feasible. Further improvements in NK cell function can be reached by inducing memory-like NK cells, developing chimeric antigen receptor (CAR)-NK cells, or isolating NK-cell-based tumor-infiltrating lymphocytes (TILs). Memory-like NK cells demonstrate higher in vivo persistence and cytotoxicity, with early clinical trials demonstrating safety and promising efficacy. Recent trials using CAR-NK cells have also demonstrated a lack of any major toxicity, including cytokine release syndrome, and, yet, promising clinical activity. Recent data support that the presence of TIL-NK cells is associated with improved overall patient survival in different types of solid tumors such as head and neck, colorectal, breast, and gastric carcinomas, among the most significant. In conclusion, this review presents insights into the diverse strategies available for NK cell expansion, including the roles played by various cytokines, feeder cells, and culture material in influencing the activation phenotype, telomere length, and cytotoxic potential of expanded NK cells. Notably, genetically modified K562 cells have demonstrated significant efficacy in promoting NK cell expansion. Furthermore, culturing NK cells with IL-2 and IL-15 has been shown to improve expansion rates, while the presence of IL-12 and IL-21 has been linked to enhanced cytotoxic function. Overall, this review provides an overview of NK cell expansion methodologies, highlighting the current landscape of clinical trials and the key advancements to enhance NK-cell-based adoptive cell therapy.

Abstract Natural killer cells are an important part of the innate immune system mediating robust responses to virus-infected and malignant cells without needing prior antigen priming. NK cells have always been thought to be short-lived and with no antigen specificity; however, recent data support the presence of NK cell memory including in the hapten-specific contact hypersensitivity model and in certain viral infections. The memory-like features can also be generated by short-term activation of both murine and human NK cells with cytokine combination of IL-12, IL-15 and IL-18, imparting increased longevity and enhanced anticancer functionality. Preclinical studies and very early clinical trials demonstrate safety and very promising clinical activity of these cytokine-induced memory-like (CIML) NK cells, making them an attractive cell type for developing novel adoptive cellular immunotherapy strategies. Furthermore, efforts are on to arm them with novel gene constructs for enhanced tumor targeting and function.

Background Responses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell–based therapy is a promising modality to treat post-HCT relapse.Methods We initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High-resolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion.Results In the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires.Conclusion Given their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies.Trial Registration ClinicalTrials.gov NCT04024761.Funding Dunkin’ Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.

CD6 is a co-stimulatory receptor expressed on T cells that binds activated leukocyte cell adhesion molecule (ALCAM), expressed on antigen presenting cells, epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T-cell activation, proliferation, and trafficking. In this study we examined expression of CD6 by reconstituting T cells in 95 patients after allogeneic cell transplantation and evaluated the effects of itolizumab, an anti- CD6 monoclonal antibody, on T-cell activation. CD6 T cells reconstituted early after transplant with CD4 regulatory T cells (Treg)-expressing lower levels of CD6 compared to conventional CD4 T cells (Tcon) and CD8 T cells. After onset of acute graft-versus-host disease (aGvHD), CD6 expression was further reduced in Treg and CD8 T cells compared to healthy donors, while no difference was observed for Tcon. ALCAM expression was highest in plasmacytoid dendritic cells (pDC), lowest in myeloid dendritic cells (mDC) and intermediate in monocytes and was generally increased after aGvHD onset. Itolizumab inhibited CD4 and CD8 T-cell activation and proliferation in preGvHD samples, but inhibition was less prominent in samples collected after aGvHD onset, especially for CD8 T cells. Functional studies showed that itolizumab did not mediate direct cytolytic activity or antibody-dependent cytotoxicity in vitro. However, itolizumab efficiently abrogated the costimulatory activity of ALCAM on T-cell proliferation, activation and maturation. Our results identify the CD6-ALCAM pathway as a potential target for aGvHD control and a phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGvHD is currently ongoing (clinicaltrials gov. Identifier: NCT03763318).

Natural killer cells constitute a part of the innate immune system that mediates an effective immune response towards virus-infected and malignant cells. In recent years, research has focused on exploring and advancing NK cells as an active immunotherapy platform. Despite major advances, there are several key challenges that need to be addressed for the effective translation of NK cell research to clinical applications. This review highlights some of these challenges and the innovative strategies being developed to overcome them, including in vitro expansion, in vivo persistence, infiltration to the tumor site, and prevention of exhaustion.

Abstract: In the absence of prospective studies that examine the effect of conditioning regimen intensity after T-cell–replete haploidentical transplant for acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and myelodysplastic syndrome (MDS), a retrospective cohort analysis was performed. Of the 1325 eligible patients (AML, n = 818; ALL, n = 286; and MDS, n = 221), 526 patients received a myeloablative regimen and 799 received a reduced-intensity regimen. Graft-versus-host disease prophylaxis was uniform with posttransplant cyclophosphamide, a calcineurin inhibitor, and mycophenolate mofetil. The primary end point was disease-free survival. Cox regression models were built to study the effect of conditioning regimen intensity on transplant outcomes. For patients aged 18 to 54 years, disease-free survival was lower (hazard ratio [HR], 1.34; 42% vs 51%; P = .007) and relapse was higher (HR, 1.51; 44% vs 33%; P = .001) with a reduced-intensity regimen compared with a myeloablative regimen. Nonrelapse mortality did not differ according to regimen intensity. For patients aged 55 to 70 years, disease-free survival (HR, 0.97; 37% vs 43%; P = .83) and relapse (HR, 1.32; 42% vs 31%; P = .11) did not differ according to regimen intensity. Nonrelapse mortality was lower with reduced-intensity regimens (HR, 0.64; 20% vs 31%; P = .02). Myeloablative regimens are preferred for AML, ALL, and MDS; reduced-intensity regimens should be reserved for those unable to tolerate myeloablation.

Abstract: HLA haploidentical hematopoietic cell transplantation (haplo-HCT) using posttransplantation cyclophosphamide (PT-Cy) is an alternative strategy when a matched sibling donor (MSD) is not available. We performed a systematic review and meta-analysis to compare the outcomes of MSD vs haplo-HCT. Eleven studies (1410 haplo-HCT and 6396 MSD recipients) were meta-analyzed. All studies were retrospective and high quality, and 9 were multicenter. Haplo-HCT was associated with ~50% lower risk of chronic graft-versus-host disease (GVHD) (hazard ratio [HR], 0.55; 95% confidence interval [CI], 0.41-0.74), but higher risk of nonrelapse mortality (HR, 1.36; 95% CI, 1.12-1.66). Relapse, survival, acute GVHD, and GVHD-free relapse-free survival were not significantly different between the groups. Deciphering the relative contribution of PT-Cy and HLA disparity to the observed outcome differences between the groups requires further research.

Abstract Background There is a paucity of data on the role of allogeneic hematopoietic cell transplantation (allo-HCT) in patients with angioimmunoblastic T-cell lymphoma (AITL). Using the CIBMTR registry, we report here the outcomes of AITL patients undergoing an allo-HCT. Methods We evaluated 249 adult AITL patients who received their first allo-HCT during 2000–2016. Results The median patient age was 56 years (range = 21–77). Majority of the patients were Caucasians (86%), with a male predominance (60%). Graft-versus-host disease (GVHD) prophylaxis was predominantly calcineurin inhibitor-based approaches while the most common graft source was peripheral blood (97%). Median follow-up of survivors was 49 months (range = 4–170 months). The cumulative incidence of grade 2–4 and grade 3–4 acute GVHD at day 180 were 36% (95% CI = 30–42) and 12 (95% CI = 8–17), respectively. The cumulative incidence of chronic GVHD at 1 year was 49% (95%CI 43–56). The 1-year non-relapse mortality (NRM) was 19% (95% CI = 14–24), while the 4-year relapse/progression, progression-free survival (PFS), and overall survival (OS) were 21% (95% CI = 16–27), 49% (95% CI = 42–56), and 56% (95% CI = 49–63), respectively. On multivariate analysis, chemoresistant status at the time of allo-HCT was associated with a significantly higher risk for therapy failure (inverse of PFS) (RR = 1.73 95% CI = 1.08–2.77), while KPS

We sought to study whether survival after haploidentical transplantation is comparable to that after matched unrelated donor transplantation for 822 patients aged 50-75 years with acute myeloid leukemia in first or second complete remission. One hundred and ninety-two patients received grafts from haploidentical donors (sibling 25%; offspring 75%) and 631 patients from matched unrelated donors aged 18-40 years. Patients’ and disease characteristics of the two groups were similar except that recipients of matched unrelated donor transplantation were more likely to have poor risk cytogenetics and more likely to receive myeloablative conditioning regimens. Time from documented remission to transplant did not differ by donor type. Five-year overall survival was 32% and 42% after haploidentical and matched unrelated donor transplant, respectively (P=0.04). Multivariable analysis showed higher mortality (hazard ratio 1.27, P=0.04) and relapse (hazard ratio 1.32, P=0.04) after haploidentical transplantation, with similar non-relapse mortality risks. Chronic graft-versus-host disease was higher after matched unrelated donor compared to haploidentical transplantation when bone marrow was the graft (hazard ratio 3.12, P

Hematopoietic stem cell transplantation from a haploidentical donor is increasingly used and has become a standard donor option for patients lacking an appropriately matched sibling or unrelated donor. Historically, prohibitive immunological barriers resulting from the high degree of HLA-mismatch included graft-vs.-host disease (GVHD) and graft failure. These were overcome with increasingly sophisticated strategies to manipulate the sensitive balance between donor and recipient immune cells. Three different approaches are currently in clinical use: (a) ex vivo T-cell depletion resulting in grafts with defined immune cell content (b) extensive immunosuppression with a T-cell replete graft consisting of G-CSF primed bone marrow and PBSC (GIAC) (c) T-cell replete grafts with post-transplant cyclophosphamide (PTCy). Intriguing studies have recently elucidated the immunologic mechanisms by which PTCy prevents GVHD. Each approach uniquely affects post-transplant immune reconstitution which is critical for the control of post-transplant infections and relapse. NK-cells play a key role in haplo-HCT since they do not mediate GVHD but can successfully mediate a graft-vs.-leukemia effect. This effect is in part regulated by KIR receptors that inhibit NK cell cytotoxic function when binding to the appropriate HLA-class I ligands. In the context of an HLA-class I mismatch in haplo-HCT, lack of inhibition can contribute to NK-cell alloreactivity leading to enhanced anti-leukemic effect. Emerging work reveals immune evasion phenomena such as copy-neutral loss of heterozygosity of the incompatible HLA alleles as one of the major mechanisms of relapse. Relapse and infectious complications remain the leading causes impacting overall survival and are central to scientific advances seeking to improve haplo-HCT. Given that haploidentical donors can typically be readily approached to collect additional stem- or immune cells for the recipient, haplo-HCT represents a unique platform for cell- and immune-based therapies aimed at further reducing relapse and infections. The rapid advancements in our understanding of the immunobiology of haplo-HCT are therefore poised to lead to iterative innovations resulting in further improvement of outcomes with this compelling transplant modality.

Donor lymphocyte infusion has been used in the management of relapsed hematologic malignancies after allogeneic hematopoietic cell transplantation. It can eradicate minimal residual disease or be used to rescue a hematologic relapse, being able to induce durable remissions in a subset of patients. With the increased use of haploidentical hematopoietic cell transplantation, there is renewed interest in the use of donor lymphocytes to either treat or prevent disease relapse post transplant. Published retrospective and small prospective studies have shown encouraging results with therapeutic donor lymphocyte infusion in different haploidentical transplantation platforms. In this consensus paper, finalized on behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation, we summarize the available evidence on the use of donor lymphocyte infusion from haploidentical donor, and provide recommendations on its therapeutic, pre-emptive and prophylactic use in clinical practice.

Abstract: We sought to identify whether posttransplantation cyclophosphamide (PT-Cy) reduces or eliminates the detrimental impact of HLA mismatching on outcomes of HLA-haploidentical related donor transplantation for acute leukemia. Data from 2143 donor-recipient pairs (n = 218 haploidentical sibling; n = 218 offspring; n = 1707 HLA-matched sibling) with acute myeloid or lymphoblastic leukemia were studied. All received a calcineurin inhibitor for graft-versus-host disease (GVHD) prophylaxis while high-dose PT-Cy was also given to recipients of haploidentical transplant. Patient age correlated with donor-recipient relationship: haploidentical siblings donated to patients aged 18 to 54 years whereas offspring donated to patients aged 55 to 76 years. Therefore, transplant outcomes were examined separately in the 2 patient age groups. In patients aged 18 to 54 years, there were no significant differences in outcomes except chronic GVHD, which was lower after haploidentical sibling compared to HLA-matched sibling transplant (hazard ratio [HR], 0.63; P < .001). In patients aged 55 to 76 years, despite lower chronic GVHD (HR, 0.42; P < .001), graft failure (14% vs 6%; P = .003), nonrelapse mortality (HR, 1.48; P = .02), and overall mortality (HR, 1.32; P = .003) were higher after transplant from offspring compared with an HLA-matched sibling. These data demonstrate a superior outcome in older recipients when using an HLA-matched sibling instead of offspring, although there were differences in transplant platforms (GVHD prophylaxis and graft type) between the 2 groups. Validation of these findings requires a prospective randomized trial wherein the transplant platforms can be closely matched.

Abstract: We studied the association between non-HLA donor characteristics (age, sex, donor-recipient relationship, blood group [ABO] match, and cytomegalovirus [CMV] serostatus) and transplant outcomes after T-cell-replete HLA-haploidentical transplantation using posttransplantation cyclophosphamide (PT-Cy) in 928 adults with hematologic malignancy transplanted between 2008 and 2015. Siblings (n = 358) and offspring (n = 450) were the predominant donors, with only 120 patients having received grafts from parents. Although mortality risks were higher with donors aged 30 years or older (hazard ratio, 1.39; P < .0001), the introduction of patient age to the Cox regression model negated the effect of donor age. Two-year survival adjusted for CMV seropositivity, disease, and disease risk index was lower in patients aged 55 to 78 years after transplantation of grafts from donors younger than 30 years (53%) or aged at least 30 years (46%) compared with younger patients who received grafts from donors younger than 30 years (61%) and at least 30 years (60%; P < .0001). Similarly, 2-year survival in patients aged 55 to 78 years was lower after transplantation of grafts from siblings (45%) or offspring (48%) compared with patients aged 18 to 54 years after transplantation of grafts from siblings (62%), offspring (58%), and parents (61%; P < .0001). Graft failure was higher after transplantation of grafts from parents (14%) compared with siblings (6%) or offspring (7%; P = .02). Other non-HLA donor characteristics were not associated with survival or graft failure. The current analyses suggest patient and disease, rather than non-HLA donor characteristics, predominantly influence survival in adults.

Natural killer (NK) cells are innate lymphoid cells important for host defense against pathogens and mediate antitumor immunity. Cytokine receptors transduce important signals that regulate proliferation, survival, activation status, and trigger effector functions. Here, we review the roles of major cytokines that regulate human NK cell development, survival, and function, including IL-2, IL-12, IL-15, IL-18, and IL-21, and their translation to the clinic as immunotherapy agents. We highlight a recent development in NK cell biology, the identification of innate NK cell memory, and focus on cytokine-induced memory-like (CIML) NK cells that result from a brief, combined activation with IL-12, IL-15, and IL-18. This activation results in long lived NK cells that exhibit enhanced functionality when they encounter a secondary stimulation and provides a new approach to enable NK cells for enhanced responsiveness to infection and cancer. An improved understanding of the cellular and molecular aspects of cytokine-cytokine receptor signals has led to a resurgence of interest in the clinical use of cytokines that sustain and/or activate NK cell antitumor potential. In the future, such strategies will be combined with negative regulatory signal blockade and enhanced recognition to comprehensively enhance NK cells for immunotherapy.