Strategies to reduce relapse risk in patients undergoing allogeneic stem cell transplantation for acute myeloid leukaemia.

Allogeneic stem cell transplantation is a centrally important curative strategy in adults with acute myeloid leukaemia; however, relapse occurs in a significant proportion of patients and remains the leading cause of treatment failure. The prognosis for patients who relapse post-transplant remains poor, and the development of new strategies with the ability to reduce disease recurrence without increasing transplant toxicity remains a priority. In this review, within the context of our understanding of disease biology and the graft-versus-leukaemia (GVL) effect, we will discuss established, evolving and novel approaches for increasing remission rates, decreasing measurable residual disease pretransplant, future methods to augment the GVL effect and the opportunities for post-transplant maintenance. Future progress depends upon the development of innovative trials and networks, which will ensure the rapid assessment of emerging therapies in prospective clinical trials.

PD-1 is imprinted on cytomegalovirus-specific CD4+ T cells and attenuates Th1 cytokine production whilst maintaining cytotoxicity.

PD-1 is expressed on exhausted T cells in cancer patients but its physiological role remains uncertain. We determined the phenotype, function and transcriptional correlates of PD-1 expression on cytomegalovirus-specific CD4+ T cells during latent infection. PD-1 expression ranged from 10-85% and remained stable over time within individual donors. This 'setpoint' was correlated with viral load at primary infection. PD-1+ CD4+ T cells display strong cytotoxic function but generate low levels of Th1 cytokines which is only partially reversed by PD-1 blockade. TCR clonotypes showed variable sharing between PD-1+ and PD-1- CMV-specific cells indicating that PD-1 status is defined either during T cell priming or subsequent clonal expansion. Physiological PD-1+ CD4+ T cells therefore display a unique 'high cytotoxicity-low cytokine' phenotype and may act to suppress viral reactivation whilst minimizing tissue inflammation. Improved understanding of the physiological role of PD-1 will help to delineate the mechanisms, and potential reversal, of PD-1+ CD4+ T cell exhaustion in patients with malignant disease.

Brief cycling intervals incrementally increase the number of hematopoietic stem and progenitor cells in human peripheral blood.

Introduction: Peripheral blood stem cell (PBSC) donation is the primary procedure used to collect hematopoietic stem and progenitor cells (HSPCs) for hematopoietic stem cell transplantation. Single bouts of exercise transiently enrich peripheral blood with HSPCs and cytolytic natural killer cells (CD56dim), which are important in preventing post-transplant complications. To provide a rationale to investigate the utility of exercise in a PBSC donation setting (≈3 h), this study aimed to establish whether interval cycling increased peripheral blood HSPC and CD56dim concentrations to a greater degree than continuous cycling. Methods: In a randomised crossover study design, eleven males (mean ± SD: age 25 ± 7 years) undertook bouts of moderate intensity continuous exercise [MICE, 30 min, 65%-70% maximum heart rate (HRmax)], high-volume high intensity interval exercise (HV-HIIE, 4 × 4 min, 80%-85% HRmax) and low-volume HIIE (LV-HIIE, 4 × 2 min, 90%-95% HRmax). The cumulative impact of each interval on circulating HSPC (CD34+CD45dimSSClow) and CD56dim concentrations (cells/µL), and the bone marrow homing potential of HSPCs (expression of CXCR-4 and VLA-4) were determined. Results: There was an increase in HSPC concentration after two intervals of LV-HIIE (Rest: 1.84 ± 1.55 vs. Interval 2: 2.94 ± 1.34, P = 0.01) and three intervals of HV-HIIE only (Rest: 2.05 ± 0.86 vs. Interval 3: 2.51 ± 1.05, P = 0.04). The concentration of all leukocyte subsets increased after each trial, with this greatest for CD56dim NK cells, and in HIIE vs. MICE (LV-HIIE: 4.77 ± 2.82, HV-HIIE: 4.65 ± 2.06, MICE: 2.44 ± 0.77, P < 0.0001). These patterns were observed for concentration, not frequency of CXCR-4+ and VLA-4+ HSPCs, which was unaltered. There was a marginal decrease in VLA-4, but not CXCR-4 expression on exercise-mobilised HSPCs after all trials (P < 0.0001). Discussion: The results of the present study indicate that HIIE caused a more marked increase in HSPC and CD56dim NK cell concentrations than MICE, with mobilised HSPCs maintaining their bone marrow homing phenotype. LV-HIIE evoked an increase in HSPC concentration after just 2 × 2-minute intervals. The feasibility and clinical utility of interval cycling in a PBSC donation context should therefore be evaluated.

CD70 identifies alloreactive T cells and represents a potential target for prevention and treatment of acute GVHD.

ABSTRACT: Graft-versus-host disease (GVHD) remains a major challenge after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and further understanding of its immunopathology is crucial for developing new treatments. CD70 interacts with CD27 and is upregulated transiently on T cells after recent T-cell receptor (TCR) engagement. Here, we investigated the functional and clinical significance of CD70 expression on T cells during the early posttransplantation period. CD70 was expressed on a subset of highly activated memory T cells within the first 2 weeks after transplant, which then gradually declined in most patients. CD70+ T cells exhibited an open chromatin landscape and a transcriptional profile indicative of intense Myelocytomatosis oncogene (MYC)-driven glycolysis and proliferation. CD4+ and CD8+CD70+ T-cell numbers increased by ninefold and fourfold, respectively, during acute GVHD (aGVHD) and displayed an oligoclonal TCR repertoire. These cells expressed CCR4 and CCR6 chemokine receptors and were markedly increased in aGVHD tissue samples. Furthermore, CD70+ T cells demonstrated alloreactive specificity in vitro, and proliferative and inflammatory cytokine responses were markedly attenuated by CD70 blockade. These findings identify CD70 as a marker of highly activated alloreactive T cells and reveal the potential therapeutic importance of inhibiting CD27-CD70 costimulation in both the prophylaxis and treatment of aGVHD.

Early inflammatory markers as prognostic indicators following allogeneic stem cell transplantation.

Allogeneic stem cell transplantation is used widely in the treatment of hematopoietic malignancy although graft versus host disease and relapse remain major complications. We measured the serum protein expression of 92 inflammation-related markers from 49 patients at Day 0 (D0) and 154 patients at Day 14 (D14) following transplantation and related values to subsequent clinical outcomes. Low levels of 7 proteins at D0 were linked to GvHD whilst high levels of 7 proteins were associated with relapse. The concentration of 38 proteins increased over 14 days and higher inflammatory response at D14 was strongly correlated with patient age. A marked increment in protein concentration during this period associated with GvHD but reduced risk of disease relapse, indicating a link with alloreactive immunity. In contrast, patients who demonstrated low dynamic elevation of inflammatory markers during the first 14 days were at increased risk of subsequent disease relapse. Multivariate time-to-event analysis revealed that high CCL23 at D14 was associative of AGvHD, CXCL10 with reduced rate of relapse, and high PD-L1 with reduced overall survival. This work identifies a dynamic pattern of inflammatory biomarkers in the very early post-transplantation period and reveals early protein markers that may help to guide patient management.

CD52/GPI- T-Cells Are Enriched for Alloreactive Specificity and Predict Acute Graft-Versus-Host-Disease After Stem Cell Transplantation.

Alemtuzumab is a CD52-specific lympho-depleting antibody. CD52- T cells emerge under alemtuzumab selection pressure. We sought to investigate the phenotype and function of the CD52- T cell fraction and related their presence to clinical outcome. We obtained longitudinal peripheral blood samples from 67 consecutive patients undergoing allo-HSCT between 2013-2016. Forty-seven patients (70%) had a myeloid disease (acute myelogenous leukemia or myelodysplastic syndrome) whereas 20 patients had lymphoid disease. All patients received in vivo alemtuzumab (10 mg/d from day -5 for 5 days) as part of their conditioning protocol. Sixty-three (94%) received reduced-intensity conditioning chemotherapy, whereas 4 (6%) received a myeloablative regimen. All patients received post-transplantation cyclosporine A for graft-versus-host disease (GVHD) prophylaxis. Six (9%) also received methotrexate, whereas 2 (3%) patients also received mycophenolate mofetil. Overall survival at 2 years was 68%, and relapse-free survival was 48%. Twenty-none percent of patients experienced acute GVHD (grade 2 or above), and 15% developed chronic GVHD. CD52- T cells were detectable in 66 of 67 consecutive patients. CD52- T cells demonstrated low binding of fluorescent aerolysin, indicating downregulation of the glycophosphatidylinositol anchor, although we did not detect any mutations in the PIG-A gene as is typically seen in patients with paroxysmal nocturnal hemoglobinuria. CD52- T cells were almost exclusively CD4+ and exhibited a dominant memory phenotype with only small numbers of CD25+ CD127low Foxp3+ regulatory T cells. CD52- T cells exhibited alloreactive specificity in vitro and have a distinct TCR repertoire to CD52+ T cells. Early after allo-hematopoietic stem cell transplantation, the presence of a significant population of CD52- T cells (comprising >51% of the T cell fraction) was found to be an independent risk factor for acute GvHD. This was confirmed in a validation cohort of 28 patients obtained between 2017-2018. These data suggest that the CD52- T cell fraction may represent a residual "footprint" of an early CD4+ T cell alloreactive response and may have been rescued from alemtuzumab-mediated lysis by antigen engagement in vivo. These data help to delineate the nature of T cell escape from alemtuzumab surveillance and contribute to increasing interest in the importance of CD4+ T cells in alloreactive immune responses, which could help inform immunotherapy protocols.

Mixed chimerism established by hematopoietic stem cell transplantation is maintained by host and donor T regulatory cells.

Transplantation is an effective treatment of many clinical disorders, but the mechanisms that regulate immunological tolerance are uncertain and remain central to improving patient outcome. Hemopoietic stem cell transplantation (SCT) often establishes "mixed chimerism" in which immune cells from both the donor and patient coexist in vivo in a setting of immunological tolerance. We studied immune function in 69 patients within 2 months following SCT; 37 were fully donor and 32 displayed mixed chimerism. The proportion of T regulatory (Treg) cells was increased during mixed chimerism and comprised equal numbers of donor and host-derived regulatory cells. This was associated with a tolerogenic PD-L1+ profile on dendritic cells. Importantly, effector T cells from patients with mixed chimerism exhibited reduced cytotoxicity against host target cells in vitro, but this was restored following depletion of CD4+ Treg cells. These data show that Treg cells play a major role in sustaining immunological tolerance during mixed chimerism. These insights should help to guide novel interventions to improve clinical transplantation.

Very early lineage-specific chimerism after reduced intensity stem cell transplantation is highly predictive of clinical outcome for patients with myeloid disease.

BACKGROUND: The importance of chimerism status in the very early period after hematopoietic stem cell transplantation is unclear. We determined PBMC and T-cell donor chimerism 50 days after transplantation and related this to disease relapse and overall survival. METHODS: 144 sequential patients underwent transplantation of which 90 had AML/MDS and 54 had lymphoma. 'Full donor chimerism' was defined as ≥99% donor cells and three patient groups were defined: 40% with full donor chimerism (FC) in both PBMC and T-cells; 25% with mixed chimerism (MC) within both compartments and 35% with 'split' chimerism (SC) characterised by full donor chimerism within PBMC and mixed chimerism within T-cells. RESULTS: In patients with myeloid disease a pattern of mixed chimerism (MC) was associated with a one year relapse rate of 45% and a five year overall survival of 40% compared to values of 8% and 75%, and 17% and 60%, for those with SC or FC respectively. The pattern of chimerism had no impact on clinical outcome for lymphoma. CONCLUSION: The pattern of lineage-specific chimerism at 50 days after transplantation is highly predictive of clinical outcome for patients with myeloid malignancy and may help to guide subsequent clinical management.

Donor-derived mycosis fungoides following reduced intensity haematopoietic stem cell transplantation from a matched unrelated donor.

A 46-year-old woman with a history of dasatinib-resistant chronic myeloid leukaemia, clonal evolution and monosomy 7 underwent reduced intensity conditioned in vivo T-cell-depleted allogeneic haematopoietic stem cell transplantation (HSCT) from a matched unrelated donor. Following the transplantation, she developed recurrent cutaneous graft versus host disease (GvHD), which required treatment with systemic immunosuppression and electrocorporeal photophoresis. Concurrently, she developed a lichenoid rash with granulomatous features suggestive of cutaneous sarcoidosis. Additional treatment with hydroxychloroquine was initially successful, but 2 months later, she developed erythroderma with palpable lymphadenopathy. Repeated histological analysis established a diagnosis of folliculotropic mycosis fungoides stage IVA2, and the malignant clone was confirmed to be of donor origin. A positive response to brentuximab has been shown. This is the first reported case of primary mycosis fungoides after matched unrelated donor HSCT, and in a patient still undergoing treatment for GvHD.

Establishment and functioning of intrathymic microenvironments.

The thymus supports the production of self-tolerant T cells from immature precursors. Studying the mechanisms regulating the establishment and maintenance of stromal microenvironments within the thymus therefore is essential to our understanding of T-cell production and ultimately immune system functioning. Despite our ability to phenotypically define stromal cell compartments of the thymus, the mechanisms regulating their development and the ways by which they influence T-cell precursors are still unclear. Here, we review recent findings and highlight unresolved issues relating to the development and functioning of thymic stromal cells.