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The diagnostic spectrum for AML patients is increasingly based on genetic abnormalities due to their prognostic and predictive value. However, information on the AML blast phenotype regarding their maturational arrest has started to regain importance due to its predictive power for drug responses. Here, we deconvolute 1350 bulk RNA-seq samples from five independent AML cohorts on a single-cell healthy BM reference and demonstrate that the morphological differentiation stages (FAB) could be faithfully reconstituted using estimated cell compositions (ECCs). Moreover, we show that the ECCs reliably predict ex-vivo drug resistances as demonstrated for Venetoclax, a BCL-2 inhibitor, resistance specifically in AML with CD14+ monocyte phenotype. We validate these predictions using LUMC proteomics data by showing that BCL-2 protein abundance is split into two distinct clusters for NPM1-mutated AML at the extremes of CD14+ monocyte percentages, which could be crucial for the Venetoclax dosing patients. Our results suggest that Venetoclax resistance predictions can also be extended to AML without recurrent genetic abnormalities and possibly to MDS-related and secondary AML. Lastly, we show that CD14+ monocytic dominated Ven/Aza treated patients have significantly lower overall survival. Collectively, we propose a framework for allowing a joint mutation and maturation stage modeling that could be used as a blueprint for testing sensitivity for new agents across the various subtypes of AML., (© 2024. The Author(s).)

Introduction: Unmodified donor lymphocyte infusions (DLI) after allogeneic stem cell transplantation (alloSCT) can boost the beneficial Graft-versus-Leukemia (GvL) effect but may also induce severe Graft-versus-Host-Disease (GvHD). To improve the balance between GvL and GvHD, it is crucial to identify factors that influence the alloreactivity of DLI., Methods: We investigated the effects of the presence of patient-derived antigen-presenting cells at time of DLI as estimated by the bone marrow (BM) chimerism status, lymphopenia as measured by the absolute lymphocyte count (ALC) at time of DLI, and the presence of a viral infection ( de novo or reactivation) close to DLI on the risk of GvHD after DLI. The cohort consisted of patients with acute leukemia or myelodysplastic syndrome who prophylactically or pre-emptively received DLI as standard care after alemtuzumab-based alloSCT. In patients at high risk for relapse, DLI was administered at 3 months after alloSCT (n=88) with a dose of 0.3x10 6 or 0.15x10 6 T cells/kg in case of a related or unrelated donor, respectively. All other patients (n=76) received 3x10 6 or 1.5x10 6 T cells/kg, respectively, at 6 months after alloSCT., Results: For both DLIs, patients with reduced-intensity conditioning and an unrelated donor had the highest risk of GvHD. For DLI given at three months, viral infection within 1 week before and 2 weeks after DLI was an additional significant risk factor (hazard ratio (HR) 3.66 compared to no viral infection) for GvHD. At six months after alloSCT, viral infections were rare and not associated with GvHD. In contrast, mixed BM chimerism (HR 3.63 for ≥5% mixed chimerism compared to full donor) was an important risk factor for GvHD after DLI given at six months after alloSCT. ALC of <1000x10 6 /l showed a trend for association with GvHD after this DLI (HR 2.05 compared to ≥1000x106/l, 95% confidence interval 0.94-4.45). Furthermore, the data suggested that the presence of a viral infection close to the DLI at three months or ≥5% mixed chimerism at time of the DLI at six months correlated with the severity of GvHD, thereby increasing their negative impact on the current GvHD-relapse-free survival., Conclusion: These data demonstrate that the risk factors for GvHD after DLI depend on the setting of the DLI., 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., (Copyright © 2024 Koster, von dem Borne, van Balen, Marijt, Tjon, Snijders, van Lammeren, Veelken, Falkenburg, Halkes and de Wreede.)

Survival after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for severe idiopathic aplastic anemia (SAA) has improved in recent years, approaching 75% at 5 years. However, an SAA-adapted composite endpoint, graft-versus-host disease (GvHD) and relapse/rejection-free survival (GRFS), may more accurately assess patient outcomes beyond survival. We analyzed GRFS to identify risk factors and specific causes of GRFS failure. Our retrospective analysis from the Severe Aplastic Anemia Working Party of the European Society for Blood and Marrow Transplantation included 479 patients with idiopathic SAA who underwent allo-HSCT in two conventional situations: i) upfront allo-HSCT from a matched related donor (MRD) (upfront cohort), and ii) allo-HSCT for relapsed or refractory SAA (rel/ref cohort). Relevant events for GRFS calculation included graft failure, grade 3-4 acute GvHD, extensive chronic GvHD, and death. In the upfront cohort (n=209), 5-year GRFS was 77%. Late allo-HSCT (i.e., >6 months after SAA diagnosis) was the main poor prognostic factor, specifically increasing the risk of death as the cause of GRFS failure (hazard ratio [HR]=4.08; 95% confidence interval [CI]: 1.41-11.83; P=0.010). In the rel/ref cohort (n=270), 5-year GRFS was 61%. Age was the main factor significantly increasing the risk of death (HR=1.04; 95% CI: 1.02-1.06; P<0.001), acute GvHD (HR=1.03; 95% CI: 1.00-1.07; P=0.041), and chronic GvHD (HR=1.04; 95% CI: 1.01-1.08; P=0.032) as the cause of GRFS failure. GRFS after upfront MRD allo-HSCT was very good, notably with early allo-HSCT, confirming that younger patients with an MRD should be transplanted immediately. GRFS was worse in cases of salvage allo-HSCT, most notably in older patients, questioning the utility of allo-HSCT earlier in the disease course.

Alloreactive donor-derived T-cells play a pivotal role in alloimmune responses after allogeneic hematopoietic stem cell transplantation (alloSCT); both in the relapse-preventing Graft-versus-Leukemia (GvL) effect and the potentially lethal complication Graft-versus-Host-Disease (GvHD). The balance between GvL and GvHD can be shifted by removing T-cells via T-cell depletion (TCD) to reduce the risk of GvHD, and by introducing additional donor T-cells (donor lymphocyte infusions [DLI]) to boost the GvL effect. However, the association between T-cell kinetics and the occurrence of allo-immunological events has not been clearly demonstrated yet. Therefore, we investigated the complex associations between the T-cell kinetics and alloimmune responses in a cohort of 166 acute leukemia patients receiving alemtuzumab-based TCD alloSCT. Of these patients, 62 with an anticipated high risk of relapse were scheduled to receive a prophylactic DLI at 3 months after transplant. In this setting, we applied joint modelling which allowed us to better capture the complex interplay between DLI, T-cell kinetics, GvHD and relapse than traditional statistical methods. We demonstrate that DLI can induce detectable T-cell expansion, leading to an increase in total, CD4+ and CD8+ T-cell counts starting at 3 months after alloSCT. CD4+ T-cells showed the strongest association with the development of alloimmune responses: higher CD4 counts increased the risk of GvHD (hazard ratio 2.44, 95% confidence interval 1.45-4.12) and decreased the risk of relapse (hazard ratio 0.65, 95% confidence interval 0.45-0.92). Similar models showed that natural killer cells recovered rapidly after alloSCT and were associated with a lower risk of relapse (HR 0.62, 95%-CI 0.41-0.93). The results of this study advocate the use of joint models to further study immune cell kinetics in different settings., 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., (Copyright © 2023 Koster, Bonneville, Borne, van Balen, Marijt, Tjon, Snijders, van Lammeren, Veelken, Putter, Falkenburg, Halkes and de Wreede.)

After allogeneic stem cell transplantation (alloSCT), patient-derived stem cells that survived the pretransplantation conditioning compete with engrafting donor stem cells for bone marrow (BM) repopulation. In addition, donor-derived alloreactive T cells present in the stem cell product may favor establishment of complete donor-derived hematopoiesis by eliminating patient-derived lymphohematopoietic cells. T cell-depleted alloSCT with sequential transfer of potentially alloreactive T cells by donor lymphocyte infusion (DLI) provides a unique opportunity to selectively study how competitive repopulation and allo-immunologic pressure influence lymphohematopoietic recovery. This study aimed to determine the relative contribution of competitive repopulation and donor-derived anti-recipient alloimmunologic pressure on the establishment of lymphohematopoietic chimerism after alloSCT. In this retrospective cohort study of 281 acute leukemia patients treated according to a protocol combining alemtuzumab-based T cell-depleted alloSCT with prophylactic DLI, we investigated engraftment and quantitative donor chimerism in the BM and immune cell subsets. DLI-induced increase of chimerism and development of graft-versus-host disease (GVHD) were analyzed as complementary indicators for donor-derived anti-recipient alloimmunologic pressure. Profound suppression of patient immune cells by conditioning sufficed for sustained engraftment without necessity for myeloablative conditioning or development of clinically significant GVHD. Although 61% of the patients without any DLI or GVHD showed full donor chimerism (FDC) in the BM at 6 months after alloSCT, only 24% showed FDC in the CD4 + T cell compartment. In contrast, 75% of the patients who had received DLI and 83% of the patients with clinically significant GVHD had FDC in this compartment. In addition, 72% of the patients with mixed hematopoiesis receiving DLI converted to complete donor-derived hematopoiesis, of whom only 34% developed clinically significant GVHD. Our data show that competitive repopulation can be sufficient to reach complete donor-derived hematopoiesis, but that some alloimmunologic pressure is needed for the establishment of a completely donor-derived T cell compartment, either by the development of GVHD or by administration of DLI. We illustrate that it is possible to separate the graft-versus-leukemia effect from GVHD, as conversion to durable complete donor-derived hematopoiesis following DLI did not require induction of clinically significant GVHD., (Copyright © 2023 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Clofarabine (CLO) is a nucleoside analog with efficacy in relapsed/refractory acute lymphoblastic leukemia (ALL). This randomized phase 3 study aimed to evaluate whether CLO added to induction and whether consolidation would improve outcome in adults with newly diagnosed ALL. Treatment of younger (18-40 years) patients consisted of a pediatric-inspired protocol, and for older patients (41-70 years), a semi-intensive protocol was used. Three hundred and forty patients were randomized. After a median follow-up of 70 months, 5-year event-free survival (EFS) was 50% and 53% for arm A and B (CLO arm). For patients ≤40 years, EFS was 58% vs 65% in arm A vs B, whereas in patients >40 years, EFS was 43% in both arms. Complete remission (CR) rate was 89% in both arms and similar in younger and older patients. Minimal residual disease (MRD) was assessed in 200 patients (60%). Fifty-four of 76 evaluable patients (71%) were MRD- after consolidation 1 in arm A vs 75/81 (93%) in arm B (P = .001). Seventy (42%) patients proceeded to allogeneic hematopoietic stem cell transplantation in both arms. Five-year overall survival (OS) was similar in both arms: 60% vs 61%. Among patients achieving CR, relapse rates were 28% and 24%, and nonrelapse mortality was 16% vs 17% after CR. CLO-treated patients experienced more serious adverse events, more infections, and more often went off protocol. This was most pronounced in older patients. We conclude that, despite a higher rate of MRD negativity, addition of CLO does not improve outcome in adults with ALL, which might be due to increased toxicity. This trial was registered at www.trialregister.nl as #NTR2004., (© 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.)

Idiopathic acquired aplastic anemia can be successfully treated with Anti Thymocyte Globulin (ATG)-based immune suppressive therapy and is therefore considered a T cell-mediated auto immune disease. Based on this finding, several other forms of idiopathic acquired bone marrow failure are treated with ATG as well. For this review, we extensively searched the present literature for evidence that ATG can lead to enduring remissions in different forms of acquired multi- or single-lineage bone marrow failure. We conclude that ATG-based therapy can lead to an enduring hematopoietic response and increased overall survival (OS) in patients with acquired aplastic aplasia. In patients with hypocellular myelodysplastic syndrome, ATG can lead to a hematological improvement without changing the OS. ATG seems less effective in acquired single-lineage failure diseases like Pure Red Cell Aplasia, Amegakaryocytic Thrombocytopenia and Pure White Cell Aplasia, suggesting a different pathogenesis in these bone marrow failure states compared to aplastic anemia. T cell depletion is hypothesized to play an important role in the beneficial effect of ATG but, as ATG is a mixture of polyclonal antibodies binding to different antigens, other anti-inflammatory or immunomodulatory effects could play a role as well.

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.)

Background Aims: To reduce the risk of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT), T-cell depletion (TCD) of grafts can be performed by the addition of alemtuzumab (ALT) "to the bag" (in vitro) before transplantation. In this prospective study, the authors analyzed the effect of in vitro incubation with 20 mg ALT on the composition of grafts prior to graft infusion. Furthermore, the authors assessed whether graft composition at the moment of infusion was predictive for T-cell reconstitution and development of GVHD early after TCD alloSCT., Methods: Sixty granulocyte colony-stimulating factor-mobilized stem cell grafts were obtained from ≥9/10 HLA-matched related and unrelated donors. The composition of the grafts was analyzed by flow cytometry before and after in vitro incubation with ALT. T-cell reconstitution and incidence of severe GVHD were monitored until 12 weeks after transplantation., Results: In vitro incubation of grafts with 20 mg ALT resulted in an initial median depletion efficiency of T-cell receptor (TCR) α/β T cells of 96.7% (range, 63.5-99.8%), followed by subsequent depletion in vivo. Graft volumes and absolute leukocyte counts of grafts before the addition of ALT were not predictive for the efficiency of TCR α/β T-cell depletion. CD4 pos T cells were depleted more efficiently than CD8 pos T cells, and naive and regulatory T cells were depleted more efficiently than memory and effector T cells. This differential depletion of T-cell subsets was in line with their reported differential CD52 expression. In vitro depletion efficiencies and absolute numbers of (naive) TCR α/β T cells in the grafts after ALT incubation were not predictive for T-cell reconstitution or development of GVHD post- alloSCT., Conclusions: The addition of ALT to the bag is an easy, fast and generally applicable strategy to prevent GVHD in patients receiving alloSCT after myeloablative or non-myeloablative conditioning because of the efficient differential depletion of donor-derived lymphocytes and T cells., (Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

We provide a long-term evaluation of patients enrolled in the EORTC/GIMEMA AML-10 trial which included a total of 2157 patients, 15-60 years old, randomized to receive either daunorubicin (DNR, 50 mg/m 2 ), mitoxantrone (MXR, 12 mg/m 2 ), or idarubicin (IDA, 10 mg/m 2 ) in addition to standard-dose cytarabine and etoposide for induction chemotherapy and intermediate dose cytarabine for consolidation. Younger patients who reached complete remission with complete (CR) or incomplete (CRi) recovery were then scheduled to receive an allogeneic hematopoietic stem cell transplantation (HSCT). That was if they had a HLA-identical sibling donor; in all other cases, an autologous HSCT had to be administered. At an 11-year median follow-up, the 5-year, 10-year and 15-year overall survival (OS) rates were 33.2%, 30.1% and 28.0%, respectively. No significant difference between the three randomized groups regarding OS was observed (P = .38). In young patients, 15-45 years old, no treatment difference (P = .89) regarding OS was observed, while in patients 46-60 years old, MXR and IDA groups had a trend for a longer OS as compared to the DNR group (P = .029). Among younger patients without a favorable MRC cytogenetic risk subgroup who achieved a CR/CRi after induction chemotherapy, those with a HLA-identical sibling donor had higher 10-year and 15-year OS rates than those without. In older patients who reached CR/CRi, the long-term outcomes of those with or without a donor was similar. In conclusion, long-term outcomes of the study confirmed similar OS in the three randomized groups in the whole cohort of patients., (© 2020 Wiley Periodicals, Inc.)

There have been sporadic reports of the development of delayed disease recurrence after bone marrow transplantation for severe aplastic anemia despite sustained majority or full donor chimerism. This is termed "donor-type aplasia" (DTA). We describe the management and outcome of 11 pediatric patients from 8 institutions in Europe, the United States, and the Middle East who developed DTA at a mean of 35 months post-transplant. These patients were initially transplanted at a mean age of 10.0 years (range, 5.8 to 16.0 years), 9 from matched sibling donors and 2 from matched unrelated donors. Attempts to treat DTA with varying combinations of additional immunosuppression (including intravenous immunoglobulin, donor lymphocyte infusions, stem cell boosts, and other therapies) failed. Ten patients have received a conditioned second transplant, 9 from the same donor and 1 from a new matched unrelated donor. Aplasia has resolved in the remaining patient in response to ongoing eltrombopag therapy. All patients were alive at a mean of 92 months (range, 26 to 195) after a second transplant; 6 are in complete remission, but 4 suffered from second/recurrent DTA at 16 to 129 months after retransplant and required further transplant therapy., (Copyright © 2019 American Society for Transplantation and Cellular Therapy. All rights reserved.)

Monosomal karyotype confers a poor prognosis in patients with acute myeloid leukemia. Here, we determined the impact of the type of remission-induction chemotherapy and the impact of having a donor in younger acute myeloid leukemia patients with a monosomal karyotype included in two phase III trials. In the first trial patients were randomized to receive either daunorubicin, mitoxantrone, or idarubicin in addition to standard-dose cytarabine and etoposide for induction chemotherapy. In the second trial patients were randomized to standard-dose cytarabine or high-dose cytarabine induction, both with daunorubicin and etoposide. In both trials, patients who achieved a complete remission with or without complete hematologic recovery underwent allogeneic hematopoietic stem cell transplantation if they had a donor; otherwise, they underwent autologous transplantation. In comparison to patients with intermediate-risk cytogenetics without a monosomal karyotype (n=1,584) and with adverse cytogenetics without a monosomal karyotype (n=218), patients with a monosomal karyotype (n=188) were more likely not to achieve a complete remission with or without count recovery [odds ratio=2.85, 95% confidence interval (95%, CI): 2.10-3.88] and had shorter overall survival [hazard ratio, (HR)=2.44, 95% CI: 2.08-2.88]. There was no impact of the type of anthracycline or of the dose of cytarabine on outcomes in patients with a monosomal karyotype. Among monosomal karyo type patients who achieved a complete remission with or without count recovery, HLA-identical related donor availability was associated with longer survival from complete remission with or without count recovery (HR=0.59, 95% CI: 0.37-0.95). ClinicalTrials.gov identifiers: AML-10: NCT00002549; AML-12: NCT00004128., (Copyright© 2019 Ferrata Storti Foundation.)

Transplantation of hematopoietic cells from a healthy individual (allogeneic hematopoietic cell transplantation (allo-HCT)) demonstrates that adoptive immunotherapy can cure blood cancers: still, post-transplantation relapses remain frequent. To explain their drivers, we analyzed the genomic and gene expression profiles of acute myeloid leukemia (AML) blasts purified from patients at serial time-points during their disease history. We identified a transcriptional signature specific for post-transplantation relapses and highly enriched in immune-related processes, including T cell costimulation and antigen presentation. In two independent patient cohorts we confirmed the deregulation of multiple costimulatory ligands on AML blasts at post-transplantation relapse (PD-L1, B7-H3, CD80, PVRL2), mirrored by concomitant changes in circulating donor T cells. Likewise, we documented the frequent loss of surface expression of HLA-DR, -DQ and -DP on leukemia cells, due to downregulation of the HLA class II regulator CIITA. We show that loss of HLA class II expression and upregulation of inhibitory checkpoint molecules represent alternative modalities to abolish AML recognition from donor-derived T cells, and can be counteracted by interferon-γ or checkpoint blockade, respectively. Our results demonstrate that the deregulation of pathways involved in T cell-mediated allorecognition is a distinctive feature and driver of AML relapses after allo-HCT, which can be rapidly translated into personalized therapies.

The major cause of death after allogeneic Hematopoietic Stem Cell Transplantation (HSCT) for acute myeloid leukemia (AML) is disease relapse. We investigated the expression of Inhibitory Receptors (IR; PD-1/CTLA-4/TIM-3/LAG-3/2B4/KLRG1/GITR) on T cells infiltrating the bone marrow (BM) of 32 AML patients relapsing (median 251 days) or maintaining complete remission (CR; median 1 year) after HSCT. A higher proportion of early-differentiated Memory Stem (T SCM ) and Central Memory BM-T cells express multiple IR in relapsing patients than in CR patients. Exhausted BM-T cells at relapse display a restricted TCR repertoire, impaired effector functions and leukemia-reactive specificities. In 57 patients, early detection of severely exhausted (PD-1 + Eomes + T-bet - ) BM-T SCM predicts relapse. Accordingly, leukemia-specific T cells in patients prone to relapse display exhaustion markers, absent in patients maintaining long-term CR. These results highlight a wide, though reversible, immunological dysfunction in the BM of AML patients relapsing after HSCT and suggest new therapeutic opportunities for the disease.

Adult B-lymphoblastic leukemia (B-ALL) is a hematological malignancy characterized by genetic heterogeneity. Despite successful remission induction with classical chemotherapeutics and novel targeted agents, enduring remission is often hampered by disease relapse due to outgrowth of a pre-existing subclone resistant against the treatment. In this study, we show that small glycophosphatidylinositol (GPI)-anchor deficient CD52-negative B-cell populations are frequently present already at diagnosis in B-ALL patients, but not in patients suffering from other B-cell malignancies. We demonstrate that the GPI-anchor negative phenotype results from loss of mRNA expression of the PIGH gene, which is involved in the first step of GPI-anchor synthesis. Loss of PIGH mRNA expression within these B-ALL cells follows epigenetic silencing rather than gene mutation or deletion. The coinciding loss of CD52 membrane expression may contribute to the development of resistance to alemtuzumab (ALM) treatment in B-ALL patients resulting in the outgrowth of CD52-negative escape variants. Additional treatment with 5-aza-2'-deoxycytidine may restore expression of CD52 and revert ALM resistance., (© 2018 The Authors. American Journal of Hematology published by Wiley Periodicals, Inc.)

We retrospectively analyzed the outcomes of 1837 adults and children with severe aplastic anemia (SAA) who underwent matched sibling donor (MSD) and matched unrelated donor (MUD) hemopoietic stem cell transplantation (HSCT) between 2000 and 2013. Patients were grouped by transplant conditioning containing either anti-thymocyte globulin (ATG) (n = 1283), alemtuzumab (n = 261), or no serotherapy (NS) (n = 293). The risks of chronic GvHD were significantly reduced when ATG or alemtuzumab were compared with NS (P = .021 and .003, respectively). Acute GVHD was significantly reduced in favor of alemtuzumab compared with ATG (P = .012) and NS (P < .001). By multivariate analysis, when compared with ATG, alemtuzumab was associated with a lower risk of developing acute (OR 0.262; 95% CI 0.14-0.47; P < .001) and chronic GVHD (HR 0.58; 95% CI 0.35-0.94; P = .027). OS was significantly better in ATG and alemtuzumab patients compared with NS (P = .010 and .025). Our data shows inclusion of serotherapy in MSD and MUD HSCT for patients with SAA reduces chronic GVHD and provides a survival advantage over patients not receiving serotherapy. Notably, alemtuzumab reduced the risk of acute and chronic GvHD compared with ATG and indicates that alemtuzumab might be the serotherapy of choice for MSD and MUD transplants for SAA., (© 2018 Wiley Periodicals, Inc.)

Under non-inflammatory conditions HLA class II is predominantly expressed on hematopoietic cells. Therefore, donor CD4 T-cells after allogeneic stem cell transplantation (alloSCT) may mediate graft-vs.-leukemia reactivity without graft-vs.-host disease (GVHD). We analyzed immune responses in four patients converting from mixed to full donor chimerism without developing GVHD upon purified CD4 donor lymphocyte infusion (DLI) from their HLA-identical sibling donor after T-cell depleted alloSCT. In vivo activated T-cells were clonally isolated after CD4 DLI. Of the alloreactive T-cell clones, 96% were CD4 positive, illustrating the dominant role of CD4 T-cells in the immune responses. We identified 9 minor histocompatibility antigens (MiHA) as targets for alloreactivity, of which 8 were novel HLA class II restricted MiHA. In all patients, MiHA specific CD4 T-cells were found that were capable to lyse hematopoietic cells and to recognize normal and malignant cells. No GVHD was induced in these patients. Skin fibroblasts forced to express HLA class II, were recognized by only two MiHA specific CD4 T-cell clones. Of the 7 clones that failed to recognize fibroblasts, two targeted MiHA were encoded by genes not expressed in fibroblasts, presentation of one MiHA was dependent on HLA-DO, which is absent in fibroblasts, and T-cells recognizing the remaining 4 MiHA had an avidity that was apparently too low to recognize fibroblasts, despite clear recognition of hematopoietic cells. In conclusion, purified CD4 DLI from HLA-identical sibling donors can induce conversion from mixed to full donor chimerism with graft-vs.-malignancy reactivity, but without GVHD, by targeting HLA class II restricted MiHA.