Expanding the repertoire reveals recurrent, cryptic, and hematopoietic HLA class I minor histocompatibility antigens.

ABSTRACT: Allogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 Epstein-Barr virus transformed B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, that is translated from unconventional open reading frames, for example long noncoding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT.

Eltrombopag Added to Immunosuppression in Severe Aplastic Anemia.

BACKGROUND: A single-group, phase 1-2 study indicated that eltrombopag improved the efficacy of standard immunosuppressive therapy that entailed horse antithymocyte globulin (ATG) plus cyclosporine in patients with severe aplastic anemia. METHODS: In this prospective, investigator-led, open-label, multicenter, randomized, phase 3 trial, we compared the efficacy and safety of horse ATG plus cyclosporine with or without eltrombopag as front-line therapy in previously untreated patients with severe aplastic anemia. The primary end point was a hematologic complete response at 3 months. RESULTS: Patients were assigned to receive immunosuppressive therapy (Group A, 101 patients) or immunosuppressive therapy plus eltrombopag (Group B, 96 patients). The percentage of patients who had a complete response at 3 months was 10% in Group A and 22% in Group B (odds ratio, 3.2; 95% confidence interval [CI], 1.3 to 7.8; P = 0.01). At 6 months, the overall response rate (the percentage of patients who had a complete or partial response) was 41% in Group A and 68% in Group B. The median times to the first response were 8.8 months (Group A) and 3.0 months (Group B). The incidence of severe adverse events was similar in the two groups. With a median follow-up of 24 months, a karyotypic abnormality that was classified as myelodysplastic syndrome developed in 1 patient (Group A) and 2 patients (Group B); event-free survival was 34% and 46%, respectively. Somatic mutations were detected in 29% (Group A) and 31% (Group Β) of the patients at baseline; these percentages increased to 66% and 55%, respectively, at 6 months, without affecting the hematologic response and 2-year outcome. CONCLUSIONS: The addition of eltrombopag to standard immunosuppressive therapy improved the rate, rapidity, and strength of hematologic response among previously untreated patients with severe aplastic anemia, without additional toxic effects. (Funded by Novartis and others; RACE ClinicalTrials.gov number, NCT02099747; EudraCT number, 2014-000363-40.).

Quality of life after immune suppressive therapy in aplastic anemia.

Acquired aplastic anemia (AA) is a rare form of immune-mediated bone marrow failure, which can result in life-threatening infections or bleeding if left untreated. Treatment consists of either immune suppressive therapy (IST) or allogeneic stem cell transplantation (alloHSCT). While considerable research has been published regarding survival, response rate and toxicity of both treatments, knowledge on the impact on quality of life (QoL) is scarce. We used the recently developed AA-specific QoL questionnaire (QLQ-AA/PNH-54) to evaluate QoL in a single center cohort of AA patients who were successfully treated with IST. The 54 questions represent 12 different QoL domains. Results were analyzed for all patients and grouped based on hematologic response (complete response (CR) or partial response (PR)). Thirty-six successfully treated adult patients (15 in CR, 21 in PR) completed the questionnaire (median age 54 years, range 21-71; median time since last IST 5 years, range 0-41). Fatigue was experienced by 83% of patients. Even though total QoL scores did not significantly differ between patients with PR and CR (105 vs 92, p-value 0,17) there appeared to be a trend towards higher scores in patients with PR, especially in domains concerning psychological wellbeing. This trend was most clear in the domains fear of progression (2,12 in PR patients vs 1,73 in CR patients; p-value 0,08) and role functioning (2,22 vs 1,88; p-value 0,07). In conclusion, patients with AA continue to experience psychological and physical effects despite successful IST.

Graft-versus-host disease and relapse/rejection-free survival after allogeneic transplantation for idiopathic severe aplastic anemia: a comprehensive analysis from the SAAWP of the EBMT.

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.

Effect of alemtuzumab-based T-cell depletion on graft compositional change in vitro and immune reconstitution early after allogeneic stem cell transplantation.

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/ß 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/ß T-cell depletion. CD4pos T cells were depleted more efficiently than CD8pos 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/ß 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.

Relapse of Aplastic Anemia with Majority Donor Chimerism (Donor-Type Aplasia) Occurring Late after Bone Marrow Transplantation.

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.

Impact of the type of anthracycline and of stem cell transplantation in younger patients with acute myeloid leukaemia: Long-term follow up of a phase III study.

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/m2 ), mitoxantrone (MXR, 12 mg/m2 ), or idarubicin (IDA, 10 mg/m2 ) 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.

High Mutation Frequency of the PIGA Gene in T Cells Results in Reconstitution of GPI Anchor-/CD52- T Cells That Can Give Early Immune Protection after Alemtuzumab-Based T Cell-Depleted Allogeneic Stem Cell Transplantation.

Alemtuzumab (ALM) is used for T cell depletion in the context of allogeneic hematopoietic stem cell transplantation (alloSCT) to prevent acute graft-versus-host disease and graft rejection. Following ALM-based T cell-depleted alloSCT, relatively rapid recovery of circulating T cells has been described, including T cells that lack membrane expression of the GPI-anchored ALM target Ag CD52. We show, in a cohort of 89 human recipients of an ALM-based T cell-depleted alloSCT graft, that early lymphocyte reconstitution always coincided with the presence of large populations of T cells lacking CD52 membrane expression. In contrast, loss of CD52 expression was not overt within B cells or NK cells. We show that loss of CD52 expression from the T cell membrane resulted from loss of GPI anchor expression caused by a highly polyclonal mutational landscape in the PIGA gene. This polyclonal mutational landscape in the PIGA gene was also found in CD52- T cells present at a low frequency in peripheral blood of healthy donors. Finally, we demonstrate that the GPI-/CD52- T cell populations that arise after ALM-based T cell-depleted alloSCT contain functional T cells directed against multiple viral targets that can play an important role in immune protection early after ALM-based T cell-depleted transplantation.

Impact of induction regimen and allogeneic hematopoietic cell transplantation on outcome in younger adults with acute myeloid leukemia with a monosomal karyotype.

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.

Use of eltrombopag in aplastic anemia in Europe.

Eltrombopag (ELT), an oral thrombopoietin receptor agonist, has recently emerged as a promising new drug for the treatment of aplastic anemia (AA). How ELT is used outside of clinical trials in the real-world setting and results of this treatment are not known. We conducted therefore a retrospective survey on the use of ELT in AA among EBMT member centers. We analyzed the 134 patients reported in our survey together with 46 patients recently published by Lengline et al. The median follow-up from start of ELT treatment was 15.3 months, with 85.6% patients alive at last follow-up. Importantly, only 28.9% of our patients received ELT according to the FDA/EMA label as monotherapy in the relapsed/refractory setting, whereas 16.7% received ELT upfront. The overall response rate in our cohort was 62%, very similar to the results of the pivotal ELT trial. In multivariate analysis, combination therapy with ELT/cyclosporine/ATG and response to previous therapy were associated with response. Overall survival was favorable with a 1-year survival from ELT start of 87.4%. We identified age, AA severity before ELT start and response to ELT as variables significantly associated with OS. Two patients transformed to MDS; other adverse events were mostly benign. In sum, ELT is used widely in Europe to treat AA patients, mostly in the relapsed/refractory setting. Response to ELT is similar to the clinical trial data across different age groups, treatment lines, and treatment combinations and results in favorable survival.

Loss of the GPI-anchor in B-lymphoblastic leukemia by epigenetic downregulation of PIGH expression.

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.

Impact of T-cell depletion strategies on outcomes following hematopoietic stem cell transplantation for idiopathic aplastic anemia: A study on behalf of the European blood and marrow transplant severe aplastic anemia working party.

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.

Transplant results in adults with Fanconi anaemia.

The outcomes of adult patients transplanted for Fanconi anaemia (FA) have not been well described. We retrospectively analysed 199 adult patients with FA transplanted between 1991 and 2014. Patients were a median of 16 years of age when diagnosed with FA, and underwent transplantation at a median age of 23 years. Time between diagnosis and transplant was shortest (median 2 years) in those patients who had a human leucocyte antigen identical sibling donor. Fifty four percent of patients had bone marrow (BM) failure at transplantation and 46% had clonal disease (34% myelodysplasia, 12% acute leukaemia). BM was the main stem cell source, the conditioning regimen included cyclophosphamide in 96% of cases and fludarabine in 64%. Engraftment occurred in 82% (95% confidence interval [CI] 76-87%), acute graft-versus-host disease (GvHD) grade II-IV in 22% (95% CI 16-28%) and the incidence of chronic GvHD at 96 months was 26% (95% CI 20-33). Non-relapse mortality at 96 months was 56% with an overall survival of 34%, which improved with more recent transplants. Median follow-up was 58 months. Patients transplanted after 2000 had improved survival (84% at 36 months), using BM from an identical sibling and fludarabine in the conditioning regimen. Factors associated with improved outcome in multivariate analysis were use of fludarabine and an identical sibling or matched non-sibling donor. Main causes of death were infection (37%), GvHD (24%) and organ failure (12%). The presence of clonal disease at transplant did not significant impact on survival. Secondary malignancies were reported in 15 of 131 evaluable patients.

Cytogenetic clonal heterogeneity is not an independent prognosis factor in 15-60-year-old AML patients: results on 1291 patients included in the EORTC/GIMEMA AML-10 and AML-12 trials.

The presence of cytogenetic clonal heterogeneity has been associated with poor prognosis in patients with acute myeloid leukemia (AML). Here, we reassessed this association. The study cohort consisted of all patients with an abnormal karyotype randomized in the EORTC/GIMEMA AML-10 and AML-12 trials. Abnormal karyotypes were classified as no subclones present (cytogenetic abnormality in a single clone), defined subclones present (presence of one to three subclones), and composite karyotypes (CP) (clonal heterogeneity not allowing enumeration of individual subclones). The main endpoints were overall survival (OS) and disease-free survival (DFS). Among 1291 patients with an abnormal karyotype, 1026 had no subclones, 226 at least 1 subclone, and 39 a CP. Patients with defined subclones had an OS similar to those with no subclones (hazard ratio (HR) 1.05, 95% confidence interval (CI) 0.88-1.26), but CP patients had a shorter OS (HR = 1.58, 95% CI 1.11-2.26). However, in a multivariate Cox model stratified by protocol and adjusted for age, cytogenetic risk group, secondary versus primary AML, and performance status, clonal heterogeneity lost its prognostic importance (HR = 1.10, 95% CI 0.91-1.32 for defined subclones versus no subclones; HR = 0.96, 95% CI 0.67-1.38 for CP versus no subclones). Also, the impact of having a donor on DFS was similar in the three clonal subgroups. In summary, in patients with cytogenetic abnormality, presence of subclones had no impact on OS. The dismal outcome in patients with a CP was explained by the known predictors of poor prognosis. TRIAL REGISTRATION: AML-10: ClinicalTrials.gov identifier: NCT00002549, retrospectively registered July 19, 2004; AML12: ClinicalTrials.gov identifier: NCT00004128, registered January 27, 2003.

Characterization of acute myeloid leukemia based on levels of global hydroxymethylation.

Patients with acute myeloid leukemia (AML) frequently harbor mutations in genes involved in the DNA (hydroxy)methylation pathway (DNMT3A, TET2, IDH1, and IDH2). In this study, we measured 5-hydroxymethylcytosine (5hmC) levels in 206 clinically and molecularly well-characterized younger adult AML patients (≤60 years) included in the European Organization for Research and Treatment of Cancer/Gruppo Italiano Malattie Ematologiche dell'Adulto (EORTC/GIMEMA) AML-12 06991 clinical trial and correlated the 5hmC levels with mutational status and overall survival (OS). In healthy control cells, 5hmC levels were confined to a narrow range (1.5-fold difference), whereas in AML cells, a much wider range was detected (15-fold difference). We identified 3 5hmC subpopulations in our patient cohort (low, intermediate, and high). The low 5hmC group consisted almost entirely of patients with TET2 or IDH mutations. As expected, TET2 and IDH mutated patients had significantly lower levels of 5hmC compared with patients without mutated TET2 and IDH1/2 (both P < .001). Interestingly, high 5hmC levels correlated with inferior OS (high vs intermediate 5hmC: P = .047, hazard ratio [HR] = 1.81). Multivariate analysis revealed that high 5hmC is an independent poor prognostic indicator for OS (high vs intermediate 5hmC: P = .01, HR = 2.10). This trial was registered at www.clinicaltrials.gov as NCT00004128.

Multi-state analysis illustrates treatment success after stem cell transplantation for acute myeloid leukemia followed by donor lymphocyte infusion.

In the field of hematopoietic stem cell transplantation, the common approach is to focus outcome analyses on time to relapse and death, without assessing the impact of post-transplant interventions. We investigated whether a multi-state model would give insight into the events after transplantation in a cohort of patients who were transplanted using a strategy including scheduled donor lymphocyte infusions. Seventy-eight consecutive patients who underwent myeloablative T-cell depleted allogeneic stem cell transplantation for acute myeloid leukemia or myelodysplastic syndrome were studied. We constructed a multi-state model to analyze the impact of donor lymphocyte infusion and graft-versus-host disease on the probabilities of relapse and non-relapse mortality over time. Based on this model we introduced a new measure for outcome after transplantation which we called 'treatment success': being alive without relapse and immunosuppression for graft-versus-host disease. All relevant clinical events were implemented into the multi-state model and were denoted treatment success or failure (either transient or permanent). Both relapse and non-relapse mortality were causes of failure of comparable magnitude. Whereas relapse was the dominant cause of failure from the transplantation state, its rate was reduced after graft-versus-host disease, and especially after donor lymphocyte infusion. The long-term probability of treatment success was approximately 40%. This probability was increased after donor lymphocyte infusion. Our multi-state model helps to interpret the impact of post-transplantation interventions and clinical events on failure and treatment success, thus extracting more information from observational data.

HLA class II upregulation during viral infection leads to HLA-DP-directed graft-versus-host disease after CD4+ donor lymphocyte infusion.

CD8+ T cell-depleted (TCD) donor lymphocyte infusion (DLI) after TCD allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with a reduced risk of graft-versus-host disease (GVHD) while preserving conversion to donor hematopoiesis and antitumor immunity, providing a rationale for exploring CD4+ T cell-based immunotherapy for hematologic malignancies. Here, we analyzed the clinical course and specificity of T cell immune responses in 2 patients with acute myeloid leukemia (AML) who converted to full-donor chimerism but developed severe acute GVHD after prophylactic CD4+ DLI after 10/10-HLA-matched, but HLA-DPB1-mismatched TCD-alloSCT. Clonal analysis of activated T cells isolated during GVHD demonstrated allo-reactivity exerted by CD4+ T cells directed against patient-mismatched HLA-DPB1 molecules on hematopoietic cells and skin-derived fibroblasts only when cultured under inflammatory conditions. At the time of CD4+ DLI, both patients contained residual patient-derived T cells, including cytomegalovirus (CMV)-specific T cells as a result of CMV reactivations. Once activated by CMV antigens, these CMV-specific T cells could stimulate HLA-DPB1-specific CD4+ T cells, which in turn could target nonhematopoietic tissues in GVHD. In conclusion, our data demonstrate that GVHD after HLA-DPB1-mismatched CD4+ DLI can be mediated by allo-reactive HLA-DPB1-directed CD4+ T cells and that ongoing viral infections inducing HLA class II expression on nonhematopoietic cells may increase the likelihood of GVHD development. This trial is registered at http://www.controlled-trials.com/ISRCTN51398568/LUMC as #51398568.