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There is little long-term outcome data on the efficacy of autologous hematopoietic stem cell transplantation (ASCT) in light chain deposition disease (LCDD). We identified 51 LCDD patients in the European Society for Blood and Bone Marrow transplantation registry who had undergone upfront ASCT between 1995 and 2021. The median serum creatinine was 280 μmol/L and 45% required renal replacement therapy (RRT) at time of transplant. The melphalan dose was 100 mg/m2 in 23%, 140 mg/m2 in 55% and 200 mg/m2 in 21%. The rate of very good partial response or better improved from 41% pretransplant to 66% at day +100 post- ASCT. In RRT-independent patients, there was a modest improvement in renal function within the first 3 months; the median estimated glomerular filtration rate increased from 44 to 51 mL/min/1.73 m2. There was no further change between 3 and 12 months post-ASCT. No patient who was RRT-independent at ASCT became RRT dependent by day + 100 post-ASCT. Median follow- up post-ASCT was 84 months (interquartile range [IQR]: 46-122). At 6-years post ASCT, overall survival was 88% (95% confidence interval [CI]: 78-98) and PFS was 44% (95% CI: 28-60). The 2-year cumulative incidence of relapse and non-relapse mortality was 17% (95% CI: 6-27) and 2% (95% CI: 0-6), respectively. The cumulative incidence of renal transplantation at 4 years after ASCT was 27% (95% CI: 13-41) with renal transplantation performed between 6.3 and 52.9 months post-ASCT (median 24.7 months). ASCT represents a feasible option for LCDD patients even if RRT dependent at time of transplant. Outcomes are favorable with low non-relapse mortality and good long-term overall survival.

Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond., (© 2024. Springer Nature Limited.)

We present an immunocompromised patient with a multiresistant herpes simplex virus-1 reactivation with a rare mutation (A605V) in the viral DNA polymerase gene. Next-generation sequencing suggests the presence of multiple drug-resistant strains before treatment and altered ratios during treatment, affecting the clinical response to aciclovir and foscarnet., Competing Interests: Potential conflicts of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Therapy-related myeloid neoplasms (t-MN), either myelodysplastic neoplasms (t-MDS) or acute myeloid leukemias (t-AML), have a poor prognosis and allogeneic haematopoietic cell transplantation (allo-HCT) represents the only curative option. In this multicenter, registry-based study, we analyzed outcomes of 378 patients undergoing first allo-HCT between 2006-2017 for t-MN arising secondary to lymphoma treatment. Median age was 58 years at allo-HCT; 222 (59%) had a diagnosis of t-MDS and 156 (41%) of t-AML, respectively. At the time of allo-HCT, 46% of t-MN cases were reported as in complete remission (CR) and 15% of lymphomas were recorded as not in remission. A reduced intensity conditioning regimen was used in 70% of cases. For the entire cohort, 5-year OS, and t-MN PFS, relapse incidence and NRM were 32%, 28%, 35% and 37%, respectively. In multivariable analysis, undergoing allo-HCT with t-MN not in CR and older age were associated with significantly worse OS, PFS and NRM. At 5 years post allo-HCT, the relapse incidence of lymphoma was low at 3%, while the rate of secondary malignancies was 8%. This analysis shows the curative potential of allo-HCT for patients with t-MN arising secondary to lymphoma treatment in approximately a third of patients., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Few cancers can be targeted efficiently by engineered T cell strategies. Here, we show that γδ T cell antigen receptor (γδ TCR)-mediated cancer metabolome targeting can be combined with targeting of cancer-associated stress antigens (such as NKG2D ligands or CD277) through the addition of chimeric co-receptors. This strategy overcomes suboptimal γ9δ2 TCR engagement of αβ T cells engineered to express a defined γδ TCR (TEGs) and improves serial killing, proliferation and persistence of TEGs. In vivo, the NKG2D-CD28 WT chimera enabled control only of liquid tumors, whereas the NKG2D-4-1BB CD28TM chimera prolonged persistence of TEGs and improved control of liquid and solid tumors. The CD277-targeting chimera (103-4-1BB) was the most optimal co-stimulation format, eradicating both liquid and solid tumors. Single-cell transcriptomic analysis revealed that NKG2D-4-1BB CD28TM and 103-4-1BB chimeras reprogram TEGs through NF-κB. Owing to competition with naturally expressed NKG2D in CD8 + TEGs, the NKG2D-4-1BB CD28TM chimera mainly skewed CD4 + TEGs toward adhesion, proliferation, cytotoxicity and less exhausted signatures, whereas the 103-4-1BB chimera additionally shaped the CD8 + subset toward a proliferative state., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

We investigated whether secondary versus de novo acute myeloid leukaemia (AML) would be associated with poor outcomes in adult acute AML patients in first complete remission (CR1) receiving unrelated cord blood transplantation (CBT). This is a retrospective study from the acute leukaemia working party of the European Society for Blood and Marrow Transplantation. Inclusion criteria included adult at first allogeneic haematopoietic cell transplantation between 2000 and 2021, unrelated single or double unit CBT, AML in CR1, no ex vivo T-cell depletion and no post-transplant cyclophosphamide. The primary end-point of the study was leukaemia-free survival (LFS). A total of 879 patients with de novo (n = 696) or secondary (n = 183) AML met the inclusion criteria. In multivariable analyses, sAML patients had non-significantly different LFS (HR = 0.98, p = 0.86), overall survival (HR = 1.07, p = 0.58), relapse incidence (HR = 0.74, p = 0.09) and non-relapse mortality (HR = 1.26, p = 0.13) than those with de novo AML. Our results demonstrate non-significantly different LFS following CBT in adult patients with secondary versus de novo AML., (© 2023 British Society for Haematology and John Wiley & Sons Ltd.)

Lymphodepletion (LD) or conditioning is an essential step in the application of currently used autologous and allogeneic chimeric antigen receptor T-cell (CAR-T) therapies as it maximizes engraftment, efficacy and long-term survival of CAR-T. Its main modes of action are the depletion and modulation of endogenous lymphocytes, conditioning of the microenvironment for improved CAR-T expansion and persistence, and reduction of tumor load. However, most LD regimens provide a broad and fairly unspecific suppression of T-cells as well as other hematopoietic cells, which can also lead to severe side effects, particularly infections. We reviewed 1271 published studies (2011-2023) with regard to current LD strategies for approved anti-CD19 CAR-T products for large B cell lymphoma (LBCL). Fludarabine (Flu) and cyclophosphamide (Cy) (alone or in combination) were the most commonly used agents. A large number of different schemes and combinations have been reported. In the respective schemes, doses of Flu and Cy (range 75-120mg/m2 and 750-1.500mg/m2) and wash out times (range 2-5 days) differed substantially. Furthermore, combinations with other agents such as bendamustine (benda), busulfan or alemtuzumab (for allogeneic CAR-T) were described. This diversity creates a challenge but also an opportunity to investigate the impact of LD on cellular kinetics and clinical outcomes of CAR-T. Only 21 studies explicitly investigated in more detail the influence of LD on safety and efficacy. As Flu and Cy can potentially impact both the in vivo activity and toxicity of CAR-T, a more detailed analysis of LD outcomes will be needed before we are able to fully assess its impact on different T-cell subsets within the CAR-T product. The T2EVOLVE consortium propagates a strategic investigation of LD protocols for the development of optimized conditioning regimens., Competing Interests: MH and ML: inventors on patents related to CAR T- cell therapy filed by the University of Würzburg. MH is listed as an inventor on patent applications and granted patents related to CAR-T technologies that have been filed by the Fred Hutchinson Cancer Research Center, Seattle, WA and by the University of Würzburg, Würzburg, Germany. MH is a co-founder and equity owner of T-CURX GmbH, Würzburg, Germany. MH received honoraria from Celgene/BMS, Janssen, Kite/Gilead. VO-M: Travel grants: Kite, Celgene-BMS, Novartis, Roche, Takeda & Janssen; Consultant or advisory fees: Kite, Celgene-BMS, Miltenyi Biomedicine, Pfizer, Novartis & Janssen; Honoraria: Kite, Celgene-BMS & Janssen; Employment: Hospital Clínic de Barcelona. UJ: consultant and advisory fees from Novartis, BMS, Gilead, Janssen, Roche, Honoraria from Miltenyi Biomedicine. DB: research projects with Juno/BMS and Repairon Immuno GmbH; consultant and advisory fees from BMS and Immatics. JD: Employment: Hospital Clínic de Barcelona. DH is an employee and shareholder of Bayer AG. HN and RT: are employees of Servier IRIS. PB: Advisory Board and consultancy from Allogene, Amgen, BMS/Celgene, Kite/Gilead, Incyte, Miltenyi Biomedicine, Novartis, Nektar, Pfizer and Pierre FabreKite/Gilead, Incyte, Miltenyi Biomedicine, Novartis, Nektar, Pfizer and Pierre Fabre. JK: Received grants form Novartis, Miltenyi Biotech and Gadeta. JK is inventor and receives revenues on multiple patents on CAR T and TEG engineering. LC was employed by Takeda. AK was employed by ITTM S.A. The remaining 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., (Copyright © 2023 Lickefett, Chu, Ortiz-Maldonado, Warmuth, Barba, Doglio, Henderson, Hudecek, Kremer, Markman, Nauerth, Negre, Sanges, Staber, Tanzi, Delgado, Busch, Kuball, Luu and Jäger.)

In the setting of a first relapse of multiple myeloma (MM), a second autologous stem cell transplant (ASCT) following carfilzomib-lenalidomide-dexamethasone (KRd) is an option, although there is scarce data concerning this approach. We performed a retrospective study involving 22 EBMT-affiliated centers. Eligible MM patients had received a second-line treatment with KRd induction followed by a second ASCT between 2016 and 2018. Primary objective was to estimate progression-free survival (PFS) and overall survival (OS). Secondary objectives were to assess the response rate and identify significant variables affecting PFS and OS. Fifty-one patients were identified, with a median age of 62 years. Median PFS after ASCT was 29.5 months while 24- and 36-months OS rates were 92.1% and 84.5%, respectively. Variables affecting PFS were an interval over four years between transplants and the achievement of a very good partial response (VGPR) or better before the relapse ASCT. Our study suggests that a relapse treatment with ASCT after KRd induction is an effective strategy for patients with a lenalidomide-sensitive first relapse. Patients with at least four years of remission after a frontline ASCT and who achieved at least a VGPR after KRd induction appear to benefit the most from this approach., (© 2023. The Author(s).)

Second transplantation (HSCT2) is a potential treatment for primary graft failure (pGF). We assessed the outcome of HSCT2, performed between 2000 and 2021, for pGF in 243 patients with acute leukemia. Median age was 44.8 years. Conditioning at first HSCT (HSCT1) was myeloablative (MAC) in 58.4%. Median time from HSCT1 to HSCT2 was 48 days. Donors for HSCT2 were the same as for HSCT1 in 49%. Engraftment post HSCT2 was achieved by 73.7% of patients. The incidence of acute (a) graft versus host disease (GVHD) grades II-IV and III-IV was 23.2 and 8.1%. 5-year total and extensive chronic (c) GVHD was 22.3 and 10.1%. 5-year nonrelapse mortality (NRM), relapse incidence (RI), leukemia-free survival (LFS), overall survival (OS) and GVHD free, relapse-free survival (GRFS) was 51.6, 18.8, 29.6, 30.7 and 22.4%, respectively. Infections were the main cause of death. In multivariable analysis, being transplanted at second vs. first remission, lower Karnofsky performance status (KPS; <90) and receiving MAC at HSCT1 were adverse prognostic factors for NRM, LFS, OS, and GRFS, as was increased age for NRM, LFS, OS. We conclude that HSCT2 can rescue about a third of the patients who experienced pGF, but NRM is as high as 50%., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

γδ T cells are potent anticancer effectors with the potential to target tumours broadly, independent of patient-specific neoantigens or human leukocyte antigen background 1-5 . γδ T cells can sense conserved cell stress signals prevalent in transformed cells 2,3 , although the mechanisms behind the targeting of stressed target cells remain poorly characterized. Vγ9Vδ2 T cells-the most abundant subset of human γδ T cells 4 -recognize a protein complex containing butyrophilin 2A1 (BTN2A1) and BTN3A1 (refs. 6-8 ), a widely expressed cell surface protein that is activated by phosphoantigens abundantly produced by tumour cells. Here we combined genome-wide CRISPR screens in target cancer cells to identify pathways that regulate γδ T cell killing and BTN3A cell surface expression. The screens showed previously unappreciated multilayered regulation of BTN3A abundance on the cell surface and triggering of γδ T cells through transcription, post-translational modifications and membrane trafficking. In addition, diverse genetic perturbations and inhibitors disrupting metabolic pathways in the cancer cells, particularly ATP-producing processes, were found to alter BTN3A levels. This induction of both BTN3A and BTN2A1 during metabolic crises is dependent on AMP-activated protein kinase (AMPK). Finally, small-molecule activation of AMPK in a cell line model and in patient-derived tumour organoids led to increased expression of the BTN2A1-BTN3A complex and increased Vγ9Vδ2 T cell receptor-mediated killing. This AMPK-dependent mechanism of metabolic stress-induced ligand upregulation deepens our understanding of γδ T cell stress surveillance and suggests new avenues available to enhance γδ T cell anticancer activity., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: We have recently developed a novel T cell engager concept by utilizing γ9δ2TCR as tumor targeting domain, named gamma delta TCR anti-CD3 bispecific molecule (GAB), targeting the phosphoantigen-dependent orchestration of BTN2A1 and BTN3A1 at the surface of cancer cells. GABs are made by the fusion of the ectodomains of a γδTCR to an anti-CD3 single chain variable fragment (scFv) (γδECTO-αCD3), here we explore alternative designs with the aim to enhance GAB effectivity., Methods: The first alternative design was made by linking the variable domains of the γ and δ chain to an anti-CD3 scFv (γδVAR-αCD3). The second alternative design was multimerizing γδVAR-αCD3 proteins to increase the tumor binding valency. Both designs were expressed and purified and the potency to target tumor cells by T cells of the alternative designs was compared to γδECTO-αCD3, in T cell activation and cytotoxicity assays., Results and Discussion: The γδVAR-αCD3 proteins were poorly expressed, and while the addition of stabilizing mutations based on finding for αβ single chain formats increased expression, generation of meaningful amounts of γδVAR-αCD3 protein was not possible. As an alternative strategy, we explored the natural properties of the original GAB design (γδECTO-αCD3), and observed the spontaneous formation of γδECTO-αCD3-monomers and -dimers during expression. We successfully enhanced the fraction of γδECTO-αCD3-dimers by shortening the linker length between the heavy and light chain in the anti-CD3 scFv, though this also decreased protein yield by 50%. Finally, we formally demonstrated with purified γδECTO-αCD3-dimers and -monomers, that γδECTO-αCD3-dimers are superior in function when compared to similar concentrations of monomers, and do not induce T cell activation without simultaneous tumor engagement. In conclusion, a γδECTO-αCD3-dimer based GAB design has great potential, though protein production needs to be further optimized before preclinical and clinical testing., Competing Interests: JK reports grants from Gadeta, Novartis, and Miltenyi Biotech and is the inventor on patents dealing with γδT cell-related aspects, as well as the co-founder and shareholder of Gadeta. EvD and DB are inventor on patents dealing with γδT cell-related aspects. The remaining 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., (Copyright © 2023 van Diest, Nicolasen, Kramer, Zheng, Hernández-López, Beringer and Kuball.)

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies., (© 2022. The Author(s).)

Unmanipulated haploidentical hematopoietic stem cell transplantation (HCT) with post-transplantation cyclophosphamide as graft-versus-host disease (GVHD) prophylaxis (haplo-PTCY) and unrelated double-unit umbilical cord blood transplantation (dUCBT) are feasible options for treating patients with high-risk acute myelogenous leukemia (AML). This study compared outcomes after dUCBT and haplo-HCT using peripheral blood stem cells (PBSCs) in adult patients with AML in complete remission (CR) who underwent transplantation in European Society for Blood and Marrow Transplantation (EBMT)-affiliated centers. In a population of adults with de novo AML in first or second CR, we compared outcomes after dUCBT (n = 165) and after haplo-PTCY PBSC (n = 544) performed between January 2013 and December 2018. Patients receiving in vivo antithymocyte globulin, Campath, or ex vivo T cell depletion were excluded. The median follow-up was 33 months for the haplo-PTCY arm and 52 months for the dUCBT arm. No statistically significant differences were observed between the 2 arms in the rates of grade II-IV acute graft-versus-host disease (GVHD) (hazard ratio [HR], 1.31; P = .18), grade III-IV acute GVHD (HR, 1.17; P = .56), chronic GVHD (HR, .86; P = .48), relapse (HR, 1.07; P = .77), nonrelapse mortality (NRM) (HR, .94; P = .77), leukemia-free survival (LFS) (HR, .99; P = .95), or overall survival (OS) (HR, .99; P = .97). Favorable cytogenetic risk was the sole factor predictive of lower relapse incidence (RI). Younger age at transplantation was associated with lower NRM and higher LFS and OS. Both dUCBT and haplo-PTCY with PBSCs can be considered valid approaches for adult AML patients in CR. New strategies should be investigated in both settings to define the most appropriate conditioning regimen and potentially decrease RI and NRM through better immune reconstitution and optimal supportive care., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Allo-HSCT with CCR5Δ32/Δ32 donor cells is the only curative HIV-1 intervention. We investigated the impact of allo-HSCT on the viral reservoir in PBMCs and post-mortem tissue in two patients. IciS-05 and IciS-11 both received a CCR5Δ32/Δ32 allo-HSCT. Before allo-HSCT, ultrasensitive HIV-1 RNA quantification; HIV-1-DNA quantification; co-receptor tropism analysis; deep-sequencing and viral characterization in PBMCs and bone marrow; and post-allo-HSCT, ultrasensitive RNA and HIV-1-DNA quantification were performed. Proviral quantification, deep sequencing, and viral characterization were done in post-mortem tissue samples. Both patients harbored subtype B CCR5-tropic HIV-1 as determined genotypically and functionally by virus culture. Pre-allo-HSCT, HIV-1-DNA could be detected in both patients in bone marrow, PBMCs, and T-cell subsets. Chimerism correlated with detectable HIV-1-DNA LTR copies in cells and tissues. Post-mortem analysis of IciS-05 revealed proviral DNA in all tissue biopsies, but not in PBMCs. In patient IciS-11, who was transplanted twice, no HIV-1-DNA could be detected in PBMCs at the time of death, whereas HIV-1-DNA was detectable in the lymph node. In conclusion, shortly after CCR5Δ32/Δ32, allo-HSCT HIV-1-DNA became undetectable in PBMCs. However, HIV-1-DNA variants identical to those present before transplantation persisted in post-mortem-obtained tissues, indicating that these tissues play an important role as viral reservoirs.

γ9δ2T cells fill a distinct niche in human immunity due to the unique physiology of the phosphoantigen-reactive γ9δ2TCR. Here, we highlight reproducible TCRδ complementarity-determining region 3 (CDR3δ) repertoire patterns associated with γ9δ2T cell proliferation and phenotype, thus providing evidence for the role of the CDR3δ in modulating in vivo T-cell responses. Features that determine γ9δ2TCR binding affinity and reactivity to the phosphoantigen-induced ligand in vitro appear to similarly underpin in vivo clonotypic expansion and differentiation. Likewise, we identify a CDR3δ bias in the γ9δ2T cell natural killer receptor (NKR) landscape. While expression of the inhibitory receptor CD94/NKG2A is skewed toward cells bearing putative high-affinity TCRs, the activating receptor NKG2D is expressed independently of the phosphoantigen-sensing determinants, suggesting a higher net NKR activating signal in T cells with TCRs of low affinity. This study establishes consistent repertoire-phenotype associations and justifies stratification for the T-cell phenotype in future research on γ9δ2TCR repertoire dynamics., Competing Interests: JK reports grants from Gadeta, Novartis, and Miltenyi Biotech and is the inventor of patents dealing with γδT cell-related aspects, as well as the co-founder and shareholder of Gadeta. AV, AJ, DB, and ZS are inventors of patents dealing with γδT cell-related aspects. The remaining 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., (Copyright © 2022 Vyborova, Janssen, Gatti, Karaiskaki, Yonika, van Dooremalen, Sanders, Beringer, Straetemans, Sebestyen and Kuball.)

Graft-versus-host disease (GVHD) is the most important complication of allogeneic hematopoietic stem cell transplantation (alloHSCT). We performed a prospective randomized, multicenter, phase 3 trial to study whether posttransplant cyclophosphamide (PT-Cy) combined with a short course of cyclosporine A (CsA) would result in a reduction of severe GVHD and improvement of GVHD-free, relapse-free survival (GRFS) as compared with the combination of CsA and mycophenolic acid (MPA) after nonmyeloablative (NMA) matched related and unrelated peripheral blood alloHSCT. Between October 2013 and June 2018, 160 patients diagnosed with a high-risk hematological malignancy and having a matched related or at least 8 out of 8 HLA-matched unrelated donor were randomized and allocated in a 1:2 ratio to CsA/MPA or PT-Cy/CsA; a total of 151 patients were transplanted (52 vs 99 patients, respectively). The cumulative incidence of grade 2 to 4 acute GVHD at 6 months was 48% in recipients of CsA/MPA vs 30% following PT-Cy/CsA (hazard ratio [HR], 0.48; 95% confidence interval [CI], 0.29-0.82; P = .007). The 2-year cumulative incidence of extensive chronic GVHD was 48% vs 16% (HR, 0.36; 95% CI, 0.21-0.64; P < .001). The 1-year estimate of GRFS was 21% (11% to 32%) vs 45% (35% to 55%), P < .001. With a median follow-up of 56.4 months, relapse incidence, progression-free survival, and overall survival were not significantly different between the 2 treatment arms. PT-Cy combined with a short course of CsA after NMA matched alloHSCT significantly improves GRFS due to a significant reduction in severe acute and chronic GVHD., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Intracranial hemorrhage is seen more frequently in acute leukemia patients compared to the general population. Besides leukemia-related risk factors, also risk factors that are present in the general population might contribute to hemorrhagic complications in leukemia patients. Of those, cardiovascular risk factors leading to chronic vascular damage could modulate the occurrence of intracranial hemorrhage in these patients, as during their disease and treatment acute endothelial damage occurs due to factors like thrombocytopenia and inflammation., Objectives: Our aim was to explore if cardiovascular risk factors can predict intracranial hemorrhage in acute leukemia patients., Methods: In a case-control study nested in a cohort of acute leukemia patients, including 17 cases with intracranial hemorrhage and 55 matched control patients without intracranial hemorrhage, data on cardiovascular risk factors were collected for all patients. Analyses were performed via conditional logistic regression., Results: Pre-existing hypertension and ischemic heart disease in the medical history were associated with intracranial hemorrhage, with an incidence rate ratio of 12.9 (95% confidence interval [CI] 1.5 to 109.2) and 12.1 (95% CI 1.3 to110.7), respectively., Conclusion: Both pre-existing hypertension and ischemic heart disease seem to be strong predictors of an increased risk for intracranial hemorrhage in leukemia patients., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Chimeric antigen receptor (CAR) T cell therapies have resulted in profound clinical responses in the treatment of CD19-positive hematological malignancies, but a significant proportion of patients do not respond or relapse eventually. As an alternative to CAR T cells, T cells can be engineered to express a tumor-targeting T cell receptor (TCR). Due to HLA restriction of TCRs, CARs have emerged as a preferred treatment moiety when targeting surface antigens, despite the fact that functional differences between engineered TCR (eTCR) T and CAR T cells remain ill-defined. Here, we compared the activity of CAR T cells versus engineered TCR T cells in targeting the B cell malignancy-associated antigen CD20 as a function of antigen exposure. We found CAR T cells to be more potent effector cells, producing higher levels of cytokines and killing more efficiently than eTCR T cells in a short time frame. However, we revealed that the increase of antigen exposure significantly impaired CAR T cell expansion, a phenotype defined by high expression of coinhibitory molecules and effector differentiation. In contrast, eTCR T cells expanded better than CAR T cells under high antigenic pressure, with lower expression of coinhibitory molecules and maintenance of an early differentiation phenotype, and comparable clearance of tumor cells., Competing Interests: The authors report no conflict of interest., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The optimal CD34 + cell dose in the setting of RIC allo-HCT for myelofibrosis (MF) remains unknown. We retrospectively analyzed 657 patients with primary or secondary MF transplanted with use of peripheral blood (PB) stem cells after fludarabine/melphalan or fludarabine/busulfan RIC regimen. Median patient age was 58 (range, 22-76) years. Donors were HLA-identical sibling (MSD) or unrelated (UD). Median follow-up was 46 (2-194) months. Patients transplanted with higher doses of CD34 + cells (>7.0 × 10 6 /kg), had an increased chance of achievement of both neutrophil (hazard ratio (HR), 1.46; P < 0.001) and platelet engraftment (HR, 1.43; P < 0.001). In a model with interaction, for patients transplanted from a MSD, higher CD34 + dose was associated with improved overall survival (HR, 0.63; P = 0.04) and relapse-free survival (HR, 0.61; P = 0.02), lower risk of non-relapse mortality (HR, 0.57; P = 0.04) and higher rate of platelet engraftment. The combined effect of higher cell dose and UD was apparent only for higher neutrophil and platelet recovery rate. We did not document any detrimental effect of high CD34 + dose on transplant outcomes. More bulky splenomegaly was an adverse factor for survival, engraftment and NRM. Our analysis suggests a potential benefit for MF patients undergoing RIC PB-allo-HCT receiving more than 7.0 × 10 6 /kg CD34 + cells., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

In the complex interplay between inflammation and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-HSCT), viral reactivations are often observed and cause substantial morbidity and mortality. As toxicity after allo-HSCT within the context of viral reactivations is mainly driven by αβ T cells, we describe that by delaying αβ T cell reconstitution through defined transplantation techniques, we can harvest the full potential of early reconstituting γδ T cells to control viral reactivations. We summarize evidence of how the γδ T cell repertoire is shaped by CMV and EBV reactivations after allo-HSCT, and their potential role in controlling the most important, but not all, viral reactivations. As most γδ T cells recognize their targets in an MHC-independent manner, γδ T cells not only have the potential to control viral reactivations but also to impact the underlying hematological malignancies. We also highlight the recently re-discovered ability to recognize classical HLA-molecules through a γδ T cell receptor, which also surprisingly do not associate with GVHD. Finally, we discuss the therapeutic potential of γδ T cells and their receptors within and outside the context of allo-HSCT, as well as the opportunities and challenges for developers and for payers.

Consensus on timing of post-hematopoietic stem cell transplantation (HSCT) vaccination is currently lacking and is therefore assessed in this review. PubMed was searched systematically for articles concerning vaccination post-HSCT and included a basis in predefined criteria. To enable comparison, data were extracted and tables were constructed per vaccine, displaying vaccine response as either seroprotection or seroconversion for allogeneic HSCT (alloHSCT) and autologous HSCT (autoHSCT) separately. A total of 33 studies were included with 1914 patients in total: 1654 alloHSCT recipients and 260 autoHSCT recipients. In alloHSCT recipients, influenza vaccine at 7-48 months post-transplant resulted in responses of 10-97%. After 12 months post-transplant, responses were >45%. Pneumococcal vaccination 3-25 months post-transplant resulted in responses of 43-99%, with the response increasing with time. Diphtheria, tetanus, pertussis, poliomyelitis and Haemophilus influenzae type b at 6-17 months post-transplant: 26-100%. Meningococcal vaccination at 12 months post-transplant: 65%. Hepatitis B vaccine at 6-23 months post-transplant: 40-94%. Measles, mumps and rubella at 41-69 months post-transplant: 19-72%. In general, autoHSCT recipients obtained slightly higher responses compared with alloHSCT recipients. Conclusively, responses to childhood immunization vaccines post-HSCT are poor in comparison with healthy individuals. Therefore, evaluation of response might be indicated. Timing of revaccination is essential for optimal response. An individualized approach might be necessary for optimizing vaccine responses.

Vaccination after hematopoietic stem cell transplantation (HSCT) is essential to protect high-risk patients against potentially lethal infections. Though multiple studies have evaluated vaccine specific responses, no comprehensive analysis of a complete vaccination schedule post-HSCT has been performed and little is known about predictors for vaccine failure. In this context, allogeneic HSCT (alloHSCT) patients were included and vaccinated starting one year post-transplantation. Antibody responses were measured by Multiplex Immuno Assay for pneumococcal (PCV13), meningococcal C, diphtheria, pertussis, tetanus and Haemophilus influenza type b one month after the last vaccination and correlated to clinical and immunological parameters. Vaccine failure was defined as antibody response above vaccine-specific cut-off values for less than four out of six vaccines. Ninety-six patients were included of which 27.1% was found to have vaccine failure. Only 40.6% of all patients responded adequately to all six vaccines. In multivariate analysis, viral reactivation post-HSCT (OR 6.53; P = 0.03), B-cells <135 per mm 3 (OR 7.24; P = 0.00) and NK-cells <170 per mm 3 (OR 11.06; P = 0.00) were identified as predictors for vaccine failure for vaccination at one year post-alloHSCT. Measurement of antibody responses and an individualized approach for revaccination guided by clinical status and immune reconstitution of B-cells and NK-cells may improve vaccine responses., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Competing Interests: Competing interests: JK is shareholder of Gadeta (www.gadeta.nl). JK, ZS, DB, AV, and EvD are authors on the following patent applications: 2013: WO 2013 /147606 A1 Combinatorial gamma 9 delta 2 T cell receptor chain exchange, (C. Grunder, JK); 2017: 20170319674 Use of antibodies for enrichment of engineered t cells with exogenous immune receptors and antibodies for use in depletion of engineered t cells (JK) ; 2017: WO/2017/2017/212074 Novel method for identifying delta T cell (or gamma T cell) receptor chains or parts thereof that mediate antitumor or anti-infectious response (JK and colleagues; 2017: WO/2017/212072 Human leukocyte antigen restricted gamma delta T cell receptors (JK and colleagues), 2017/ 62508807 (ZS & JK) Composition and methods for cell targeting therapies; 2018/ 6044506 (EvD, DB, JK): GABs as next generation of immune therapy

Nonmyeloablative regimens are used for allogeneic hematopoietic cell transplantation (HCT) of older or medically unfit patients, but successful outcome is still hindered by graft-versus-host disease (GVHD), especially in the setting of HLA-mismatched HCT. New GVHD prophylaxis strategies are emerging, including the triple drug strategy, that improve the GVHD-free and relapse-free survival (GRFS). Because the impact of ATG in HLA-mismatched Flu-TBI-based nonmyeloablative HCT has not been investigated, we did a retrospective analysis in three Dutch centers. 67 patients were evaluable, with a median age of 56 years. Overall survival, relapse-free survival and GRFS at 4 years were 52%, 43%, and 38%, respectively. NRM findings and cumulative incidence of relapse at 4 years were 26% and 31%, respectively. At 1-year grade II-IV had occurred in 40% of the patients, and the incidence of moderate-severe chronic GVHD incidence was 16%. Acknowledging the limitations of retrospective analyses, we conclude that the use of ATG for HLA-mismatched truly nonmyeloablative Flu-TBI HCT is feasible and results in acceptable long term outcomes, especially with regards to GRFS. We consider ATG in combination with cyclosporin and mycophenolate mofetil as an alternative for the triple drug strategy that uses sirolimus for GVHD prophylaxis in this particular setting., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

γδT cell receptors (γδTCRs) recognize a broad range of malignantly transformed cells in mainly a major histocompatibility complex (MHC)-independent manner, making them valuable additions to the engineered immune effector cell therapy that currently focuses primarily on αβTCRs and chimeric antigen receptors (CARs). As an exception to the rule, we have previously identified a γδTCR, which exerts antitumor reactivity against HLA-A*24:02-expressing malignant cells, however without the need for defined HLA-restricted peptides, and without exhibiting any sign of off-target toxicity in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse models. This particular tumor-HLA-A*24:02-specific Vγ5Vδ1TCR required CD8αα co-receptor for its tumor reactive capacity when introduced into αβT cells engineered to express a defined γδTCR (TEG), referred to as TEG011; thus, it was only active in CD8 + TEG011. We subsequently explored the concept of additional redirection of CD4 + T cells through co-expression of the human CD8α gene into CD4 + and CD8 + TEG011 cells, later referred as TEG011&#95;CD8α. Adoptive transfer of TEG011&#95;CD8α cells in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mice injected with tumor HLA-A*24:02 + cells showed superior tumor control in comparison to TEG011, and to mock control groups. The total percentage of mice with persisting TEG011&#95;CD8α cells, as well as the total number of TEG011&#95;CD8α cells per mice, was significantly improved over time, mainly due to a dominance of CD4 + CD8 + double-positive TEG011&#95;CD8α, which resulted in higher total counts of functional T cells in spleen and bone marrow. We observed that tumor clearance in the bone marrow of TEG011&#95;CD8α-treated mice associated with better human T cell infiltration, which was not observed in the TEG011-treated group. Overall, introduction of transgenic human CD8α receptor on TEG011 improves antitumor reactivity against HLA-A*24:02 + tumor cells and further enhances in vivo tumor control., Competing Interests: DB, ZS, and JK are inventors on different patents with γδTCR sequences, recognition mechanisms, and isolation strategies. JK is cofounder and shareholder of Gadeta (www.gadeta.nl). The remaining 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., (Copyright © 2021 Johanna, Hernández-López, Heijhuurs, Scheper, Bongiovanni, de Bruin, Beringer, Oostvogels, Straetemans, Sebestyen and Kuball.)

Optimal donor choice for a second allogeneic hematopoietic cell transplant (allo-HCT) in relapsed acute lymphoblastic leukemia (ALL) remains undefined. We compared outcomes using HLA-matched unrelated donors (MUD) versus haploidentical donors in this population. Primary endpoint was overall survival (OS). The MUD allo-HCT group comprised 104 patients (male = 56, 54%), median age 36 years, mostly (76%) with B-cell phenotype in complete remission (CR) (CR2/CR3 + = 76, 73%). The 61 patients (male = 38, 62%) in the haploidentical group were younger, median age 30 years (p = 0.002), had mostly (79%) a B-cell phenotype and the majority were also in CR at time of the second allo-HCT (CR2/CR3 + = 40, 66%). Peripheral blood stem cells was the most common cell source in both, but a significantly higher number in the haploidentical group received bone marrow cells (26% vs. 4%, p < 0.0001). A haploidentical donor second allo-HCT had a 1.5-fold higher 2-year OS (49% vs. 31%), albeit not statistically significant (p = 0.13). A longer time from first allo-HCT to relapse was associated with improved OS, leukemia-free survival, graft-versus-host disease-free-relapse-free survival, and lower cumulative incidences of relapse and non-relapse mortality. Results suggest no major OS difference when choosing either a MUD or haploidentical donor for ALL patients needing a second allo-HCT., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

T cell engineering strategies offer cures to patients and have entered clinical practice with chimeric antibody-based receptors; αβT cell receptor (αβTCR)-based strategies are, however, lagging behind. To allow a more rapid and successful translation to successful concepts also using αβTCRs for engineering, incorporating a method for the purification of genetically modified T cells, as well as engineered T cell deletion after transfer into patients, could be beneficial. This would allow increased efficacy, reduced potential side effects, and improved safety of newly to-be-tested lead structures. By characterizing the antigen-binding interface of a good manufacturing process (GMP)-grade anti-αβTCR antibody, usually used for depletion of αβT cells from stem cell transplantation products, we developed a strategy that allows for the purification of untouched αβTCR-engineered immune cells by changing 2 amino acids only in the TCRβ chain constant domain of introduced TCR chains. Alternatively, we engineered an antibody that targets an extended mutated interface of 9 amino acids in the TCRβ chain constant domain and provides the opportunity to further develop depletion strategies of engineered immune cells., Competing Interests: G.J.J.K., D.X.B., Z.S., and J.K. are inventors on different patents with γδTCR sequences, recognition mechanisms, and isolation strategies. J.K. is the scientific cofounder and a shareholder of Gadeta. The remaining authors declare no competing interests., (© 2021 The Author(s).)

Various allogeneic (allo) stem cell transplantation platforms have been developed over the last 2 decades. In this review we focus on the impact of in vivo and ex vivo graft manipulation on immune reconstitution and clinical outcome. Strategies include anti-thymocyte globulin- and post-transplantation cyclophosphamide-based regimens, as well as graft engineering, such as CD34 selection and CD19/αβT cell depletion. Differences in duration of immune suppression, reconstituting immune repertoires, and associated graft-versus-leukemia effects and toxicities mediated through viral reactivations are highlighted. In addition, we discuss the impact of different reconstituting repertoires on donor lymphocyte infusions and post allo pharmacological interventions to enhance tumor control. We advocate for precisely counting all graft ingredients and therapeutic drug monitoring during conditioning in the peripheral blood, and for adjusting dosing accordingly on an individual basis. In addition, we propose novel trial designs to better assess the impact of variations in transplantation platforms in order to better learn from our diversity of "counts" and potential "adjustments." This will, in the future, allow daily clinical practice, strategic choices, and future trial designs to be based on data guided decisions, rather than relying on dogma and habits., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Background: Allogeneic hematopoietic cell transplantation (allo-HCT) using a mismatched unrelated donor (MMUD) and cord blood transplantation (CBT) are valid alternatives for patients without a fully human leukocyte antigen (HLA)-matched donor. Here, we compared the allo-HCT outcomes of CBT versus single-allele-mismatched MMUD allo-HCT with post-transplant cyclophosphamide (PTCy) in acute myeloid leukemia., Methods: Patients who underwent a first CBT without PTCy (N = 902) or allo-HCT from a (HLA 9/10) MMUD with PTCy (N = 280) were included in the study. A multivariate regression analysis was performed for the whole population. A matched-pair analysis was carried out by propensity score-based 1:1 matching of patients (177 pairs) with known cytogenetic risk., Results: The incidence of grade II-IV and grade III-IV acute graft-versus-host disease (GVHD) at 6 months was 36% versus 32% (p = 0.07) and 15% versus 11% (p = 0.16) for CBT and MMUD cohorts, respectively. CBT was associated with a higher incidence of graft failure (11% vs. 4%, p < 0.01) and higher 2-year non-relapse mortality (NRM) (30% vs. 16%, p < 0.01) compared to MMUD. In the multivariate analysis, CBT was associated with a higher risk of, NRM (HR = 2.09, 95% CI 1.46-2.99, p < 0.0001), and relapse (HR = 1.35, 95% CI 1-1.83, p = 0.05), which resulted in worse leukemia-free survival (LFS) (HR = 1.68, 95% CI 1.34-2.12, p < 0.0001), overall survival (OS) (HR = 1.7, 95% CI 1.33-2.17, p < 0.0001), and GVHD-free, relapse-free survival (GRFS) (HR = 1.49, 95% CI 1.21-1.83, p < 0.0001) compared to MMUD. The risk of grade II-IV acute GVHD (p = 0.052) and chronic GVHD (p = 0.69) did not differ significantly between the cohorts. These results were confirmed in a matched-pair analysis., Conclusions: CBT was associated with lower LFS, OS, and GRFS due to higher NRM, compared to MMUD allo-HCT with PTCy. In the absence of a fully matched donor, 9/10 MMUD with PTCy may be preferred over CBT.

Treatment results of AML in elderly patients are unsatisfactory. We hypothesized that addition of tosedostat, an aminopeptidase inhibitor, to intensive chemotherapy may improve outcome in this population. After establishing a safe dose in a run-in phase of the study in 22 patients, 231 eligible patients with AML above 65 years of age (median 70, range 66-81) were randomly assigned in this open label randomized Phase II study to receive standard chemotherapy (3+7) with or without tosedostat at the selected daily dose of 120 mg ( n = 116), days 1-21. In the second cycle, patients received cytarabine 1000 mg/m 2 twice daily on days 1-6 with or without tosedostat. CR/CRi rates in the 2 arms were not significantly different (69% (95% C.I. 60-77%) vs 64% (55-73%), respectively). At 24 months, event-free survival (EFS) was 20% for the standard arm versus 12% for the tosedostat arm (Cox-p = 0.01) and overall survival (OS) 33% vs 18% respectively ( p = 0.006). Infectious complications accounted for an increased early death rate in the tosedostat arm. Atrial fibrillation was more common in the tosedostat arm as well. The results of the present study show that the addition of tosedostat to standard chemotherapy does negatively affect the therapeutic outcome of elderly AML patients.

Lymphocyte numbers need to be quite tightly regulated. It is generally assumed that lymphocyte production and lifespan increase homeostatically when lymphocyte numbers are low and, vice versa, return to normal once cell numbers have normalized. This widely accepted concept is largely based on experiments in mice, but is hardly investigated in vivo in humans. Here we quantified lymphocyte production and loss rates in vivo in patients 0.5-1 year after their autologous hematopoietic stem cell transplantation (autoHSCT). We indeed found that the production rates of most T- and B-cell subsets in autoHSCT-patients were two to eight times higher than in healthy controls, but went hand in hand with a threefold to ninefold increase in cell loss rates. Both rates also did not normalize when cell numbers did. This shows that increased lymphocyte production and loss rates occur even long after autoHSCT and can persist in the face of apparently normal cell numbers., Competing Interests: MB, Vv, JD, Lv, AJ, SO, Mv, Rd, JB, KT No competing interests declared, JK reports grants from Novartis, Miltenyi Biotech, and Gadeta. Is inventor on multiple patents dealing with γδ T-cell research, ligands, and isolation techniques, and is scientific co-founder and shareholder of Gadeta. (Patent number: 9546998, 9891211, 10324083, 10578609. Publication number: 20200368278, 20200363397, 20190271688, 2019020961, 201901692603, 20180188234, 20170319674, 20170174741, 20150050670)., (© 2021, Baliu-Piqué et al.)

We conducted a multicenter prospective single-arm phase 1/2 study that assesses the outcome of αβ T-cell depleted allogeneic hematopoietic stem cell transplantation (allo-HSCT) of peripheral blood derived stem cells from matched related, or unrelated donors (10/10 and 9/10) in adults, with the incidence of acute graft-versus-host disease (aGVHD) as the primary end point at day 100. Thirty-five adults (median age, 59; range, 19-69 years) were enrolled. Conditioning consisted of antithymocyte globulin, busulfan, and fludarabine, followed by 28 days of mycophenolic acid after allo-HSCT. The minimal follow-up time was 24 months. The median number of infused CD34+ cells and αβ T cells were 6.1 × 106 and 16.3 × 103 cells per kg, respectively. The cumulative incidence (CI) of aGVHD grades 2-4 and 3-4 at day 100 was 26% and 14%. One secondary graft failure was observed. A prophylactic donor lymphocyte infusion (DLI) (1 × 105 CD3+ T cells per kg) was administered to 54% of the subjects, resulting in a CI of aGVHD grades 2-4 and 3-4 to 37% and 17% at 2 years. Immune monitoring revealed an early reconstitution of natural killer (NK) and γδ T cells. Cytomegalovirus reactivation associated with expansion of memory-like NK cells. The CI of relapse was 29%, and the nonrelapse mortality 32% at 2 years. The 2-year CI of chronic GVHD (cGVHD) was 23%, of which 17% was moderate. We conclude that only 26% of patients developed aGVHD 2-4 after αβ T-cell-depleted allo-HSCT within 100 days and was associated with a low incidence of cGVHD after 2 years. This trial was registered at www.trialregister.nl as #NL4767., (© 2021 by The American Society of Hematology.)

We designed a study to describe the incidence of intracranial hemorrhage according to severity and duration of thrombocytopenia and to quantify the associations of platelet transfusions with intracranial hemorrhage in patients with acute leukemia. In this case-control study nested in a cohort of 859 leukemia patients, cases (n = 17) were patients diagnosed with intracranial hemorrhage who were matched with control patients (n = 55). We documented platelet counts and transfusions for seven days before the intracranial hemorrhage in cases and in a "matched" week for control patients. Three measures of platelet count exposure were assessed in four potentially important time periods before hemorrhage. Among these leukemia patients, we observed the cumulative incidence of intracranial hemorrhage of 3.5%. Low platelet counts were, especially in the three to seven days preceding intracranial hemorrhage, associated with the incidence of intracranial hemorrhage, although with wide confidence intervals. Platelet transfusions during the week preceding the hemorrhage were associated with higher incidences of intracranial hemorrhage; rate ratios (95% confidence interval) for one or two platelet transfusions and for more than two transfusions compared with none were 4.04 (0.73 to 22.27) and 8.91 (1.53 to 51.73) respectively. Thus, among acute leukemia patients, the risk of intracranial hemorrhage was higher among patients with low platelet counts and after receiving more platelet transfusions. Especially, the latter is likely due to clinical factors leading to increased transfusion needs.

γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.

Aim: To develop a semi-mechanistic model, based on glutathione depletion and predict a previously identified intra-individual reduction in busulfan clearance to aid in more precise dosing., Methods: Busulfan concentration data, measured as part of regular care for allogeneic hematopoietic cell transplantation (HCT) patients, were used to develop a semi-mechanistic model and compare it to a previously developed empirical model. The latter included an empirically estimated time effect, where the semi-mechanistic model included theoretical glutathione depletion. As older age has been related to lower glutathione levels, this was tested as a covariate in the semi-mechanistic model. Lastly, a therapeutic drug monitoring (TDM) simulation was performed comparing the two models in target attainment., Results: In both models, a similar clearance decrease of 7% (range -82% to 44%), with a proportionality to busulfan metabolism, was found. After 40 years of age, the time effect increased with 4% per year of age (0.6-8%, P = 0.009), causing the effect to increase more than a 2-fold over the observed age-range (0-73 years). Compared to the empirical model, the final semi-mechanistic model increased target attainment from 74% to 76%, mainly through better predictions for adult patients., Conclusion: These results suggest that the time-dependent decrease in busulfan clearance may be related to gluthathione depletion. This effect increased with older age (>40 years) and was proportional to busulfan metabolism. The newly constructed semi-mechanistic model could be used to further improve TDM-guided exposure target attainment of busulfan in patients undergoing HCT., (© 2020 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Graft-versus-host disease (GVHD) and cytomegalovirus (CMV)-related complications are leading causes of mortality after unrelated-donor hematopoietic cell transplantation (UD-HCT). The non-conventional MHC class I gene MICB, alike MICA, encodes a stress-induced polymorphic NKG2D ligand. However, unlike MICA, MICB interacts with the CMV-encoded UL16, which sequestrates MICB intracellularly, leading to immune evasion. Here, we retrospectively analyzed the impact of mismatches in MICB amino acid position 98 (MICB98), a key polymorphic residue involved in UL16 binding, in 943 UD-HCT pairs who were allele-matched at HLA-A, -B, -C, -DRB1, -DQB1 and MICA loci. HLA-DP typing was further available. MICB98 mismatches were significantly associated with an increased incidence of acute (grade II-IV: HR, 1.20; 95% CI, 1.15 to 1.24; P < 0.001; grade III-IV: HR, 2.28; 95% CI, 1.56 to 3.34; P < 0.001) and chronic GVHD (HR, 1.21; 95% CI, 1.10 to 1.33; P < 0.001). MICB98 matching significantly reduced the effect of CMV status on overall mortality from a hazard ratio of 1.77 to 1.16. MICB98 mismatches showed a GVHD-independent association with a higher incidence of CMV infection/reactivation (HR, 1.84; 95% CI, 1.34 to 2.51; P < 0.001). Hence selecting a MICB98-matched donor significantly reduces the GVHD incidence and lowers the impact of CMV status on overall survival.

γδT cells play an important role in cancer immunosurveillance and are able to distinguish malignant cells from their healthy counterparts via their γδTCR. This characteristic makes γδT cells an attractive candidate for therapeutic application in cancer immunotherapy. Previously, we have identified a novel CD8α-dependent tumor-specific allo-HLA-A*24:02-restricted Vγ5Vδ1TCR with potential therapeutic value when used to engineer αβT cells from HLA-A*24:02 harboring individuals. αβT cells engineered to express this defined Vγ5Vδ1TCR (TEG011) have been suggested to recognize spatial changes in HLA-A*24:02 present selectively on tumor cells but not their healthy counterparts. However, in vivo efficacy and toxicity studies of TEG011 are still limited. Therefore, we extend the efficacy and toxicity studies as well as the dynamics of TEG011 in vivo in a humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse model to allow the preparation of a first-in-men clinical safety package for adoptive transfer of TEG011. Mice treated with TEG011 did not exhibit any graft-versus-host disease-like symptoms and extensive analysis of pathologic changes in NSG-A24:02 mice did not show any off-target toxicity of TEG011. However, loss of persistence of TEG011 in tumor-bearing mice was associated with the outgrowth of extramedullary tumor masses as also observed for mock-treated mice. In conclusion, TEG011 is well tolerated without harming HLA-A*24:02 + expressing healthy tissues, and TEG011 persistence seems to be crucial for long-term tumor control in vivo., (©2020 Society for Leukocyte Biology.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only medical intervention that has led to an HIV cure. Whereas the HIV reservoir sharply decreases after allo-HSCT, the dynamics of the T cell reconstitution has not been comprehensively described. We analyzed the activation and differentiation of CD4 + and CD8 + T cells, and the breadth and quality of HIV- and CMV-specific CD8 + T cell responses in 16 patients with HIV who underwent allo-HSCT (including five individuals who received cells from CCR5Δ32/Δ32 donors) to treat their underlying hematological malignancy and who remained on antiretroviral therapy (ART). We found that reconstitution of the T cell compartment after allo-HSCT was slow and heterogeneous with an initial expansion of activated CD4 + T cells that preceded the expansion of CD8 + T cells. Although HIV-specific CD8 + T cells disappeared immediately after allo-HSCT, weak HIV-specific CD8 + T cell responses were detectable several weeks after transplant and could still be detected at the time of full T cell chimerism, indicating that de novo priming, and hence antigen exposure, occurred during the time of T cell expansion. These HIV-specific T cells had limited functionality compared with CMV-specific CD8 + T cells and persisted years after allo-HSCT. In conclusion, immune reconstitution was slow, heterogeneous, and incomplete and coincided with de novo detection of weak HIV-specific T cell responses. The initial short phase of high T cell activation, in which HIV antigens were present, may constitute a window of vulnerability for the reseeding of viral reservoirs, emphasizing the importance of maintaining ART directly after allo-HSCT., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Nonmyeloablative (NMA) conditioning regimens facilitate allogeneic stem cell transplantation (alloSCT) in elderly patients and/or in those with comorbidities. The acute leukemia working party (ALWP) of the European Society for Blood and Marrow Transplantation (EBMT) compared the outcomes of patients ≥60 years with AML in first complete remission (CR1), that had received an alloSCT following NMA conditioning, i.e. either fludarabine/busulfan (FB2) or fludarabine/total-body-irradiation-2Gy (FluTBI2Gy). A total of 1088 patients were included (median age 65 years). Donors were matched siblings (MSD) and matched unrelated donors (MUD) in 47% and 53%, respectively. In vivo T-cell depletion (TCD) was applied to 79% and none (0%) of patients in the FB2 and FluTBI2Gy groups, respectively. In the MSD group we found a trend for less extensive cGVHD in patients receiving FB2 with in vivo TCD, HR: 0.49, p = 0.08, and in those without worse NRM, HR: 2.14, p = 0.04, and a trend for more total cGVHD, HR: 1.61, p = 0.09. Patients transplanted from MUDs had a significantly higher incidence of total cGVHD, extensive cGVHD and a worse GRFS with FluTBI2Gy in comparison to FB2, HR: 2.44; p < 0.0001; HR 4.59; p < 0.00001 and HR: 1.35; p = 0.03, respectively. No differences were observed with respect to LFS, OS, RI, NRM, and aGVHD.

γδ T cells in human solid tumors remain poorly defined. Here, we describe molecular and functional analyses of T-cell receptors (TCR) from tumor-infiltrating γδ T lymphocytes (γδ TIL) that were in direct contact with tumor cells in breast cancer lesions from archival material. We observed that the majority of γδ TILs harbored a proinflammatory phenotype and only a minority associated with the expression of IL17. We characterized TCRγ or TCRδ chains of γδ TILs and observed a higher proportion of Vδ2 + T cells compared with other tumor types. By reconstructing matched Vδ2 - TCRγ and TCRδ pairs derived from single-cell sequencing, our data suggest that γδ TILs could be active against breast cancer and other tumor types. The reactivity pattern against tumor cells depended on both the TCRγ and TCRδ chains and was independent of additional costimulation through other innate immune receptors. We conclude that γδ TILs can mediate tumor reactivity through their individual γδ TCR pairs and that engineered T cells expressing TCRγ and δ chains derived from γδ TILs display potent antitumor reactivity against different cancer cell types and, thus, may be a valuable tool for engineering immune cells for adoptive cell therapies., (©2020 American Association for Cancer Research.)