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

Objectives: Patients receiving remission induction therapy for acute myeloid leukaemia (AML) are at high risk of developing invasive fungal disease (IFD). Newer therapies with targeted antileukemic agents and the emergence of azole resistance pose a challenge to the strategy of primary antifungal prophylaxis. We report the experience of a diagnostic-driven care pathway (DCP) for the management of IFD in these patients, using only culture-directed mould inactive prophylaxis. Methods: Retrospectively, we used a single-centre study of consecutive patients receiving intensive chemotherapy for myeloid malignancies between 2014 and 2021. DCP consisted of serial cultures and serum galactomannan (sGM) screening, CT imaging, and bronchoscopy to direct targeted antifungal treatment. IFD was classified according to the 2020 EORTC/MSGERC criteria. Results: A total of 192 patients with myeloid malignancies received 300 courses of intensive chemotherapy. There were 14 cases of invasive yeast infections and 18 of probable/proven invasive mould disease (IMD). The incidence of probable/proven IMD during the first cycle of remission-induction chemotherapy was 4.6% (n = 9). sGM remained negative in all cases of invasive aspergillosis (IA), with positive mycology findings in bronchoalveolar lavage. All-cause mortality was 9.4% (n = 18) 100 days after starting chemotherapy and was comparable between patients with or without IFD. The fungal-related mortality was 1% (n = 2). Conclusion: Diagnostic-driven based management without universal mould active prophylaxis is a feasible strategy in the management of IFD and limits unnecessary antimould treatment during intensive chemotherapy. The poor performance of serial serum galactomannan screening in detecting IA warrants further investigation. [ABSTRACT FROM AUTHOR]

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

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

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

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

Background: Stem cell transplantation (SCT) is still complicated by the occurrence of fever and inflammatory complications attributed to neutropenia and subsequent infectious complications. The role of mucosal barrier injury (MBI) of the intestinal tract therein has received little attention. Methods: We performed a retrospective analysis in 163 SCT recipients of which data had been collected prospectively on intestinal damage (citrulline), inflammation (C-reactive protein), and neutrophil count. Six different conditioning regimens were studied; 5 myeloablative (MA) and 1 non-myeloablative (NMA). Linear mixed model multivariate and AUC analyses were used to define the role of intestinal damage in post-SCT inflammation. We also studied the relationship between the degree of intestinal damage and the occurrence of early post-SCT complications. Results: In the 5 MA regimen there was a striking pattern of inflammatory response that coincided with the occurrence of severe intestinal damage. This contrasted with a modest inflammatory response seen in the NMA regimen in which intestinal damage was limited. With linear mixed model analysis the degree of intestinal damage was shown the most important determinant of the inflammatory response, and both neutropenia and bacteremia had only a minor impact. AUC analysis revealed a strong correlation between citrulline and CRP (Pearson correlation r = 0.96). Intestinal damage was associated with the occurrence of bacteremia and acute lung injury, and influenced the kinetics of acute graft-versus-host disease. Conclusion: The degree of intestinal damage after myeloablative conditioning appeared to be the most important determined the inflammatory response following SCT, and was associated with inflammatory complications. Studies should explore ways to ameliorate cytotoxic therapy-induced intestinal damage in order to reduce complications associated with myeloablative conditioning therapy. [ABSTRACT FROM AUTHOR]

BACKGROUND: The use of fresh red blood cells (RBCs) is recommended for critically ill patients and patients undergoing surgery, although there is no conclusive evidence that this is beneficial. In this follow-up study, the short-term and the long-term recovery of irradiated, leukoreduced RBCs transfused after either a short storage (SS) or a long storage (LS) period were compared. By consecutive transfusion of RBCs with a SS and LS period, a direct comparison of their survival within the same patient was possible. STUDY DESIGN AND METHODS: Ten transfusion-requiring patients each received a SS RCCs (stored 0-10 days) and a LS RCCs (stored 25-35 days) consecutively. Short-term and long-term survival of the transfused RBCs was followed by flow cytometry using natural differences in RBC antigens between donors and patients. Posttransfusion recovery (PTR) was measured at several time points after transfusion. RESULTS: The mean 24-hour PTR of SS RBCs is 86.4 ± 17.8 percent and that of LS RBCs 73.5 ± 13.7 percent. After the first 24 hours, the mean times to reach a PTR of 50 percent of the 24-hour PTR (T50) and mean potential life spans (mPLs) of the surviving SS and LS RBCs (41 and 116 days and 41 and 114 days, respectively) do not differ. CONCLUSIONS: The mean 24-hour PTR of both SS and LS RBCs complies with the guidelines, even in a compromised patient population. The 24-hour PTR of SS RBCs, however, is significantly higher than that of LS RBCs. The remaining population of SS and LS RBCs has a nearly identical long-term survival. Therefore, depletion of the removal-prone RBCs before transfusion may be an efficient approach for product improvement. [ABSTRACT FROM AUTHOR]

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

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

Immune-modulating regulatory T-cell (Treg) therapy is a promising strategy in autoimmunity and transplantation. However, to achieve full clinical efficacy, better understanding of in vivo human Treg biology is warranted. Here, we demonstrate that in contrast to blood and bone marrow Tregs, which showed a resting phenotype, the majority of CD4posCD25posCD127negFoxP3pos Tregs in secondary lymphoid organs were proliferating activated CD69posCD45RAneg cells with a hyperdemethylated FOXP3 gene and a broad T-cell receptor-Vβ repertoire, implying polyclonal activation. Activated CD69pos Tregs were distributed over both T-cell and B-cell areas, distant from Airepos and CD11cpos cells. In contrast to the anergic peripheral blood Tregs, lymphoid organ Tregs had significant ex vivo proliferative capacity and produced cytokines like interleukin-2, while revealing similar suppressive potential. Also, next to Treg-expressing chemokine receptors important for a prolonged stay in lymphoid organs, a significant part of the cells expressed peripheral tissue-associated, functional homing markers. In conclusion, our data suggest that human secondary lymphoid organs aid in the maintenance and regulation of Treg function and homeostasis. This knowledge may be exploited for further optimization of Treg immunotherapy, for example, by ex vivo selection of Tregs with capacity to migrate to lymphoid organs providing an in vivo platform for further Treg expansion. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signaling could increase NK cell immune responses in high-grade EOC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Maas, Hoogstad-van Evert, Hagemans, Brummelman, van Ens, de Jonge, Hooijmaijers, Mahajan, van der Waart, Hermans, de Klein, Woestenenk, van Herwaarden, Schaap, Rezaeifard, Tauriello, Zusterzeel, Ottevanger, Jansen, Hobo and Dolstra.)

Background Aims: Natural killer (NK) cell transfer is a promising cellular immunotherapy for cancer. Previously, we developed a robust method to generate large NK cell numbers from CD34 + hematopoietic stem and progenitor cells (HSPCs), which exhibit strong anti-tumor activity. However, since these cells express low levels of the Fc receptor CD16a in vitro, antibody-dependent cellular cytotoxicity (ADCC) by these cells is limited. To broaden clinical applicability of our HSPC-NK cells toward less NK-sensitive malignancies, we aimed to improve ADCC through CD16a transduction., Methods: Using wildtype and S197P mutant greater-affinity (both with V158) CD16a retroviral transgenes (i.e., a cleavable and noncleavable CD16a upon stimulation), we generated CD16a HSPC-transduced NK cells, with CD34 + cells isolated from umbilical cord blood (UCB) or peripheral blood after G-CSF stem cell mobilization (MPB). CD16a expressing NK cells were enriched using flow cytometry-based cell sorting. Subsequently, phenotypic analyses and functional assays were performed to investigate natural cytotoxicity and ADCC activity., Results: Mean transduction efficiency was 34% for UCB-derived HSPCs and 20% for MPB-derived HSPCs, which was enriched by flow cytometry-based cell sorting to >90% for both conditions. Expression of the transgene remained stable during the entire NK expansion cell generation process. Proliferation and differentiation of HSPCs were not hampered by the transduction process, resulting in effectively differentiated CD56 + NK cells after 5 weeks. Activation of the HSPC-derived NK cells resulted in significant shedding of wildtype CD16a transcribed from the endogenous gene, but not of the noncleavable mutant CD16a protein expressed from the transduced construct. The mean increase of CD107 + IFNγ + expressing NK cells after inducing ADCC was tenfold in enriched noncleavable CD16a HSPC-NK cells. Killing capacity of CD16a-transduced NK cells was significantly improved after addition of a tumor-targeting antibody in tumor cell lines and primary B-cell leukemia and lymphoma cells compared to unmodified HSPC-NK cells., Conclusions: Together, these data demonstrate that the applicability of adoptive NK cell immunotherapy may be broadened to less NK-sensitive malignancies by upregulation of CD16a expression in combination with the use of tumor-targeting monoclonal antibodies., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: We previously reported that the "Endothelial Activation and Stress Index" (EASIX; ((creatinine×lactate dehydrogenase)÷thrombocytes)) measured before start of conditioning predicts mortality after allogeneic hematopoietic stem cell transplantation (alloSCT) when used as continuous score. For broad clinical implementation, a prospectively validated EASIX-pre cut-off is needed that defines a high-risk cohort and is easy to use., Method: In the current study, we first performed a retrospective cohort analysis in n=2022 alloSCT recipients and identified an optimal cut-off for predicting non-relapse mortality (NRM) as EASIX-pre=3. For cut-off validation, we conducted a multicenter prospective study with inclusion of n=317 first alloSCTs from peripheral blood stem cell in adult patients with acute leukemia, lymphoma or myelodysplastic syndrome/myeloproliferative neoplasms in the European Society for Blood and Marrow Transplantation network., Results: Twenty-three % (n=74) of alloSCT recipients had EASIX-pre ≥3 taken before conditioning. NRM at 2 years was 31.1% in the high EASIX group versus 11.5% in the low EASIX group (p<0.001). Patients with high EASIX-pre also had worse 2 years overall survival (51.6% vs 70.9%; p=0.002). We were able to validate the cut-off and found that EASIX ≥3 was associated with more than twofold increased risk for NRM in multivariate analysis (HR=2.18, 95% CI 1.2 to 3.94; p=0.01). No statistically significant difference could be observed for the incidence of relapse., Conclusions: The results of this study provide a prospectively validated standard laboratory biomarker index to estimate the transplant-related mortality risk after alloSCT. EASIX ≥3 taken before conditioning identifies a population of alloSCT recipients who have a more than twofold increased risk of treatment-related mortality., Competing Interests: Competing interests: OP has received honoraria or travel support from Gilead, Jazz, MSD, Novartis, Pfizer and Therakos. He has received research support from Incyte and Priothera. He is a member of advisory boards to Equillium Bio, Jazz, Gilead, Novartis, MSD, Omeros, Priothera, Sanofi, Shionogi and SOBI. HS reports having received personal fees from Incyte, Janssen, Novartis, Sanofi and from the Belgian Hematological Society (BHS), as well as research grants from Novartis and the BHS, all paid to her institution. She has also received non-financial support from Gilead, the EBMT (European Society for Blood and Marrow transplantation) and the CIBMTR (Center for International Bone Marrow Transplantation Research)., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The outcome in older patients with acute lymphoblastic leukemia (ALL) remains unsatisfactory due to high relapse and nonrelapse mortality (NRM) rates. Allogeneic stem cell transplantation (alloHSCT) as postremission therapy has an important role in reducing relapse rate, albeit its application is limited in older adult patients due to alloHSCT-related morbidity and mortality. Reduced-intensity conditioning (RIC) alloHSCT has been developed as a less toxic conditioning regimen, but comparative studies with myeloablative conditioning (MAC) are limited in patients with ALL., Methods: In this retrospective study, RIC-alloHSCT (n = 111) was compared with MAC-alloHSCT (n = 77) in patients aged 41 to 65 y with ALL in first complete remission. MAC was predominantly applied by combining high-dose total body irradiation and cyclophosphamide, whereas RIC mainly consisted of fludarabine and 2 Gy total body irradiation., Results: Unadjusted overall survival was 54% (95% confidence interval [CI], 42%-65%) at 5 y in MAC recipients compared with 39% (95% CI, 29%-49%) in RIC recipients. Overall survival and relapse-free survival were not significantly associated with type of conditioning after adjusted for the covariates age, leukemia risk status at diagnosis, donor type, and donor and recipient gender combination. NRM was significantly lower after RIC (subdistribution hazard ratio: 0.41, 95% CI, 0.22-0.78; P  = 0.006), whereas relapse was significantly higher (subdistribution hazard ratio: 3.04, 95% CI, 1.71-5.40; P < 0.001)., Conclusions: Collectively, RIC-alloHSCT has resulted in less NRM, but it was also found to be associated with a significantly higher relapse rate. These results suggest that MAC-alloHSCT may provide a more effective type of consolidation therapy for the reduction of relapse and that RIC-alloHSCT may be restricted to patients at higher risk for NRM., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The presence of TP53 mutations is associated with an unfavorable outcome in patients allografted for acute myeloid leukemia (AML), leading some to question the benefit of an allogeneic stem cell transplantation (allo-SCT) for this patient group, although this has not been studied in a large cohort., Methods: A total of 780 patients with AML in first complete remission, with either intermediate- or adverse-risk cytogenetics, whose TP53 mutation status was reported, were included in this study from the European Society for Blood and Marrow Transplantation., Results: Two-year overall survival (OS) was impaired in patients (n = 179) with evidence of a TP53 mutation at diagnosis (35.1%; 95% confidence interval [CI], 26.7-43.7) as compared to the cohort without (n = 601) (64%; 95% CI, 59.1-68.4; P = .001). In patients with mutant TP53 AML with no evidence of either chromosome 17p loss (17p-) and/or complex karyotype (CK) (n = 53, 29.6%), 2-year OS was 65.2% (95% CI, 48.4-77.6). This was not significantly different to patients without TP53 mutations. In patients with mutant TP53 AML with either 17p- and/or CK (n = 126, 70.4%), the OS was lower (24.6%; 95% CI, 16.2-34; P = .001)., Conclusions: In summary, the adverse prognostic effect of TP53 mutations in AML following an allo-SCT is not evident in patients with neither co-occurring 17p- and/or CK, and these data inform decisions regarding allo-SCT in patients with TP53 mutant AML., (© 2022 The Authors. Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Allogeneic stem cell transplantation (alloSCT), following induction chemotherapy, can be curative for hemato-oncology patients due to powerful graft-versus-tumor immunity. However, disease recurrence remains the major cause of treatment failure, emphasizing the need for potent adjuvant immunotherapy. In this regard, dendritic cell (DC) vaccination is highly attractive, as DCs are the key orchestrators of innate and adaptive immunity. Natural DC subsets are postulated to be more powerful compared with monocyte-derived DCs, due to their unique functional properties and cross-talk capacity. Yet, obtaining sufficient numbers of natural DCs, particularly type 1 conventional DCs (cDC1s), is challenging due to low frequencies in human blood. We developed a clinically applicable culture protocol using donor-derived G-CSF mobilized CD34 + hematopoietic progenitor cells (HPCs) for simultaneous generation of high numbers of cDC1s, cDC2s and plasmacytoid DCs (pDCs). Transcriptomic analyses demonstrated that these ex vivo-generated DCs highly resemble their in vivo blood counterparts. In more detail, we demonstrated that the CD141 + CLEG9A + cDC1 subset exhibited key features of in vivo cDC1s, reflected by high expression of co-stimulatory molecules and release of IL-12p70 and TNF-α. Furthermore, cDC1s efficiently primed alloreactive T cells, potently cross-presented long-peptides and boosted expansion of minor histocompatibility antigen-experienced T cells. Moreover, they strongly enhanced NK cell activation, degranulation and anti-leukemic reactivity. Together, we developed a robust culture protocol to generate highly functional blood DC subsets for in vivo application as tailored adjuvant immunotherapy to boost innate and adaptive anti-tumor immunity in alloSCT patients., (© 2021. The Author(s).)

Combining natural killer (NK) cell adoptive transfer with tumor-sensitizing chemotherapy is an attractive approach against recurrent ovarian cancer (OC), as OC is sensitive to NK cell-mediated immunity. Previously, we showed that CD34 + hematopoietic progenitor cell (HPC)-derived NK cells can kill OC cells in vitro and inhibit OC tumor growth in mice. Here, we investigated the potential of HPC-NK cell therapy combined with chemotherapeutic gemcitabine (used in recurrent OC patients) against OC. We examined the phenotypical, functional, and cytotoxic effects of gemcitabine on HPC-NK cells and/or OC cells in vitro and in OC-bearing mice. To this end, we treated OC cells and/or HPC-NK cells with or without gemcitabine and analyzed the phenotype, cytokine production, and anti-tumor reactivity. We found that gemcitabine did not affect the phenotype and functionality of HPC-NK cells, while on OC cells expression of NK cell activating ligands and death receptors was upregulated. Although gemcitabine pre-treatment of OC cells did not improve the functionality of HPC-NK cells, importantly, HPC-NK cells and gemcitabine additively killed OC cells in vitro . Similarly, combined HPC-NK cell and gemcitabine treatment additively decreased tumor growth in OC-bearing mice. Collectively, our results indicate that combination therapy of HPC-NK cells and gemcitabine results in augmented OC killing in vitro and in vivo . This provides a rationale for exploring this therapeutic strategy in patients with recurrent OC., Competing Interests: None of the authors have any competing interests., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Effective therapies for treating patients with steroid-refractory acute graft-versus-host-disease (SR-aGVHD), particularly strategies that reduce the duration of immunosuppression following remission, are urgently needed. The investigated immunotoxin combination consists of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT), which induces in vivo depletion of T cells and natural killer (NK) cells and suppresses T cell receptor activation. We conducted a phase I/II trial to examine the safety and efficacy of CD3/CD7-IT in 20 patients with SR-aGVHD; 17 of these patients (85%) had severe SR-aGVHD, and all 20 patients had visceral organ involvement, including 18 (90%) with gastrointestinal (GI) involvement and 5 (25%) with liver involvement. A validated 2-biomarker algorithm classified the majority of patients (11 of 20) as high risk. On day 28 after the start of CD3/CD7-IT therapy, the overall response rate was 60% (12 of 20), with 10 patients (50%) achieving a complete response. The 6-month overall survival rate was 60% (12 of 20), including 64% (7 of 11) classified as high risk by biomarkers. The 1-week course of treatment with CD3/CD7-IT caused profound but transient depletion of T cells and NK cells, followed by rapid recovery of the immune system with a diverse TCR Vβ repertoire, and preservation of Epstein-Barr virus- and cytomegalovirus-specific T cell clones. Furthermore, our results indicate that CD3/CD7-IT appeared to be safe and well tolerated, with a relatively low prevalence of manageable and reversible adverse events, primarily worsening of hypoalbuminemia, microangiopathy, and thrombocytopenia. These encouraging results suggest that CD3/CD7-IT may improve patient outcomes in patients with SR-aGVHD., (Copyright © 2018 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Purpose: Total-body irradiation (TBI) is a major constituent of myeloablative conditioning regimens. The standard technique consists of 12 Gy in 6 fractions over a period of 3 days. The Standard-fractionation compAred to one-daily fRaction total body irrAdiation prior to tranSplant In LEUkemia patieNts (SARASIN) study aimed to compare standard fractionation with once-daily fractionation before transplant in leukemia., Methods and Materials: We retrospectively compared TBI regimens delivered in 2993 patients from the European Society for Blood and Marrow Transplantation database, who underwent transplantation between 2000 and 2014 for acute lymphoblastic leukemia (ALL, n = 1729) or acute myeloid leukemia (AML, n = 1264). TBI was delivered as either 12 Gy in 6 fractions (group 1, considered the reference group; 1362 ALL and 857 AML patients), 9 to 12 Gy in 2 fractions (group 2, 173 ALL and 256 AML patients), or 12 Gy in 3 to 4 fractions (group 3, 194 ALL and 151 AML patients)., Results: The median follow-up was 60 and 84 months in ALL and AML patients, respectively. At 5 years, the leukemia-free survival rate, overall survival rate, relapse incidence, and nonrelapse mortality rate were 46.6%, 50.4%, 28.8%, and 24.6%, respectively, in ALL patients and 46.6%, 48.9%, 29.7%, and 23.6%, respectively, in AML patients. In multivariate analyses, the outcomes of groups 2 and 3 were not statistically different from those in group 1. The cumulative incidence of secondary malignancies (SMs) was significantly higher in group 2 (7.2%; P < 10 -6 for group 2 vs group 1). However, group 2 was not associated with an increase in SMs when we considered non-T-cell-depleted transplant patients., Conclusions: We showed that the 12-Gy fractionated TBI dose delivered either in 2 fractions or in 1 fraction per day over a period of 3 to 4 days resulted in nonsignificant differences in disease control and survival. However, 1-day fractionation may be associated with a higher risk of mucositis and hemorrhagic cystitis. The absence of a significant difference in the SM incidence in the non-T-cell-depleted group should be interpreted with caution in the context of a retrospective study design. Our findings are important to consider for radiation therapy department organization. In-depth analyses of other nonlethal toxicities and late effects are required., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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

Combining natural killer (NK) cell adoptive transfer with hypomethylating agents (HMAs) is an attractive therapeutic approach for patients with acute myeloid leukemia (AML). However, data regarding the impact of HMAs on NK cell functionality are mostly derived from in vitro studies with high nonclinical relevant drug concentrations. In the present study, we report a comparative study of azacitidine (AZA) and decitabine (DAC) in combination with allogeneic NK cells generated from CD34 + hematopoietic stem and progenitor cells (HSPC-NK cells) in in vitro and in vivo AML models. In vitro, low-dose HMAs did not impair viability of HSPC-NK cells. Furthermore, low-dose DAC preserved HSPC-NK killing, proliferation, and interferon gamma production capacity, whereas AZA diminished their proliferation and reactivity. Importantly, we showed HMAs and HSPC-NK cells could potently work together to target AML cell lines and patient AML blasts. In vivo, both agents exerted a significant delay in AML progression in NOD/SCID/IL2Rg null mice, but the persistence of adoptively transferred HSPC-NK cells was not affected. Infused NK cells showed sustained expression of most activating receptors, upregulated NKp44 expression, and remarkable killer cell immunoglobulin-like receptor acquisition. Most importantly, only DAC potentiated HSPC-NK cell anti-leukemic activity in vivo. Besides upregulation of NKG2D- and DNAM-1-activating ligands on AML cells, DAC enhanced messenger RNA expression of inflammatory cytokines, perforin, and TRAIL by HSPC-NK cells. In addition, treatment resulted in increased numbers of HSPC-NK cells in the bone marrow compartment, suggesting that DAC could positively modulate NK cell activity, trafficking, and tumor targeting. These data provide a rationale to explore combination therapy of adoptive HSPC-NK cells and DAC in patients with AML., (© 2018 by The American Society of Hematology.)

Allogeneic hematopoietic cell transplantation (HCT) offers the possibility of curative therapy for patients with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and acute myelogenous leukemia (AML). However, post-HCT relapse remains a major problem, particularly in patients with high-risk cytogenetics and in patients who cannot tolerate consolidation chemotherapy (eg, due to previous toxicity). We assessed the toxicity and efficacy of 10-day decitabine (Dec), fludarabine (Flu), and 2 Gy total body irradiation (TBI) as a new conditioning regimen for allogeneic HCT in patients with MDS, CMML, or AML. Thirty patients were enrolled, including 11 with MDS, 2 with CMML, and 17 with AML. Patients received 20 mg/m(2)/day Dec on days -11 to -2, 30 mg/m(2)/day Flu on days -4 to -2, and 2 Gy TBI on day -1, followed by infusion of a donor stem cell graft on day 0. Postgrafting immunosuppression consisted of cyclosporin A and mycophenolate mofetil. At a median follow-up of 443 days, the overall survival was 53%, relapse incidence was 27%, and nonrelapse mortality was 27%. The incidence of severe acute (grade III/IV) graft-versus-host disease (GVHD) was 27%, and that of (predominantly mild) chronic GVHD was 60%. Immunomonitoring studies revealed that specific CD8(+) T cell responses against epigenetically silenced tumor-associated antigens (TAAs), including cancer-testis antigens (MAGE-A1/A2/A3 and PRAME) and RHAMM, occurred more frequently in patients who had received Dec/Flu/TBI conditioning (8 of 11 patients) compared with a control group of patients who had received only Flu/TBI conditioning (2 of 9 patients). In summary, Dec/Flu/TBI conditioning proved feasible and effective and enhanced the induction of TAA-reactive CD8(+) T cell responses in vivo, which may contribute to disease control post-transplantation., (Copyright © 2016 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Steroid-refractory acute graft-versus-host disease (aGVHD) remains an important cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). A protocol on the management of aGVHD was introduced in our center that incorporated a prospective study on combination therapy with inolimomab (anti-IL-2Rα) and etanercept (anti-tumor necrosis factor-α) for steroid-refractory aGVHD. We evaluated the efficacy and safety in 21 consecutively treated patients. The patients had developed refractory aGVHD after SCT (n = 16) or donor lymphocyte infusion (n = 5), and aGVHD was classified as severe in all patients, mostly due to gastrointestinal involvement stages 2 to 4. No drug-related side effects were observed apart from the infections expected to occur in these severely immunocompromised patients. Overall response at day 28 of second-line therapy was 48% (10/21), with 6 and 4 patients achieving a complete and partial response, respectively. Eventually, 19 patients died (90%), with early mortality (<6 months) predominantly resulting from refractory aGVHD and secondary infections and late mortality resulting from relapse of the underlying disease. With a median follow-up of 55 days, the estimated rates of 6-month and 2-year overall survival were dismal, 29% and 10%, respectively. In conclusion, the combination of inolimomab and etanercept for steroid-refractory aGVHD failed to improve the dismal prognosis of severe steroid-refractory aGVHD., (Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Patients with chronic myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplantation may be treated by tyrosine kinase inhibitors and/or by donor lymphocyte infusions. The best strategies and timing of administration of lymphocytes are unclear. We analyzed 155 patients who relapsed after allogeneic stem cell transplantation with disease detectable only by molecular methods and who subsequently received lymphocytes. Transplants were performed in first chronic phase (n=125) or in advanced disease (n=29) from identical siblings (n=84) or unrelated donors (n=71) between 1986 and 2003. They received lymphocytes either during molecular relapse (n=85) or upon progression to more advanced disease (1993 to 2004). The median interval from relapse to lymphocyte infusion was 210 (0-1673) days. The median follow up after it was 46 (3-135) months. Overall survival was 76±4% at five years after lymphocyte infusions (89±8% with sibling donors and 63±13% with unrelated donors (P=0.003)). Survival was 69±14% when lymphocytes were given within six months of the detection of molecular relapse and 81±10% (P=0.061) when given later; 81±11% if given at molecular relapse versus 71±12% (P=0.26) with more advanced disease. In multivariate analysis survival was worse if the donor was unrelated (HR 2.54 (95% CI: 1.15-5.53), P=0.021) and better with lymphocyte infusions beyond six months from molecular relapse (HR 0.4 (95%CI: 0.19-0.84), P=0.018). These data confirm the remarkable efficacy of lymphocyte infusions for this disease. There appears to be no advantage from administering it early upon detection of molecular relapse in patients who received allogeneic stem cell transplantation for chronic myeloid leukemia., (Copyright© Ferrata Storti Foundation.)

Graft-versus-host disease (GVHD) and fungal infections are frequent complications after allogeneic hematopoietic stem cell transplantation (HSCT). Single nucleotide polymorphisms (SNPs) in genes of the immune system can influence the inflammatory cascade and T cell-driven alloimmune reactions after HSCT, and thus increasing the incidence of GVHD and infectious complications. Here, we investigated the effect of SNPs in IL-23R and CCR6 on posttransplantation outcome in 161 recipients of partially T cell-depleted HSCT. Remarkably, IL-23R SNPs were not associated with clinical outcome, but we found that disparities in the CCR6 tagSNP rs2301436 and SNP rs3093023 are independently associated with the occurrence of chronic GVHD (cGVHD) and invasive fungal disease. In multivariate analysis, patients receiving a transplant from a homozygous rs2301436 G allele donor showed less cGVHD (odds ratio [OR]: 0.16; P = .002), as was the case for a homozygous donor rs3093023 G allele (OR: 0.24; P = .005). In parallel, the GG genotype at rs2301436 in donors was associated with a higher incidence of invasive fungal disease at day 100 after HSCT (OR: 3.59; P = .008). This study shows that CCR6 SNPs can be used to predict clinical outcome, and that polymorphisms in the CCR6 gene may influence T cell-mediated immune reactions after HSCT., (Copyright © 2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Stem cell transplantation (SCT) is still complicated by the occurrence of fever and inflammatory complications attributed to neutropenia and subsequent infectious complications. The role of mucosal barrier injury (MBI) of the intestinal tract therein has received little attention., Methods: We performed a retrospective analysis in 163 SCT recipients of which data had been collected prospectively on intestinal damage (citrulline), inflammation (C-reactive protein), and neutrophil count. Six different conditioning regimens were studied; 5 myeloablative (MA) and 1 non-myeloablative (NMA). Linear mixed model multivariate and AUC analyses were used to define the role of intestinal damage in post-SCT inflammation. We also studied the relationship between the degree of intestinal damage and the occurrence of early post-SCT complications., Results: In the 5 MA regimen there was a striking pattern of inflammatory response that coincided with the occurrence of severe intestinal damage. This contrasted with a modest inflammatory response seen in the NMA regimen in which intestinal damage was limited. With linear mixed model analysis the degree of intestinal damage was shown the most important determinant of the inflammatory response, and both neutropenia and bacteremia had only a minor impact. AUC analysis revealed a strong correlation between citrulline and CRP (Pearson correlation r = 0.96). Intestinal damage was associated with the occurrence of bacteremia and acute lung injury, and influenced the kinetics of acute graft-versus-host disease., Conclusion: The degree of intestinal damage after myeloablative conditioning appeared to be the most important determined the inflammatory response following SCT, and was associated with inflammatory complications. Studies should explore ways to ameliorate cytotoxic therapy-induced intestinal damage in order to reduce complications associated with myeloablative conditioning therapy.

Background: Alloreactive NK cells play a role in tumor eradication after allogeneic HLA mismatched stem cell transplantation (SCT). The effect of NK alloreactivity in HLA identical SCT is still under debate and in particular in transplantation for chronic myeloid leukemia (CML) the data are very limited and with conflicting outcome. The aim of our study was to evaluate the effect of KIR genes and KIR ligands on leukemia free survival (LFS) and relapse rate in a well-defined, homogeneous group of CML patients phase upon HLA identical sibling SCT., Methodology: We retrospectively analyzed the effect of KIRs and KIR ligands (C1 and C2) on LFS and relapse in 70 CML patients in 1st chronic phase, who had received an HLA identical sibling graft. For KIR typing we used a single PCR based KIR typing protocol that also included primers allowing for the identification of the KIR binding site on HLA-Cw (AA 77 and 80)., Principal Findings: The data show clear differences in transplant outcome between patients having both ligands (C1 and C2) as compared to patients having only one ligand (C1 or C2). In the latter group, the stimulatory KIR2DS5 gene was associated with improved leukemia free survival (p=0.007; hazard ratio 4.3; 95% confidence interval 1.3-6.7) and lower relapse rates (p=0.028; HR 4.3, 95% CI 1.1-9.1). In contrast, in patients carrying both ligands, KIR2DS5 was associated with reduced LFS (p=0.0056; HR 0.3; 95% CI 0.1-0.7) and higher relapse rate (p=0.02; HR 0.35, 95% CI 0.1-0.8)., Conclusions: Our data indicate a role for an NK mediated anti-CML response after HLA identical sibling SCT that is influenced by KIR ligands and, more importantly, by stimulatory KIRs present in the donor.