Edward S. Morris

NKT cell–dependent leukemia eradication following stem cell mobilization with potent G-CSF analogs

NKT cells have pivotal roles in immune regulation and tumor immunosurveillance. We report that the G-CSF and FMS-like tyrosine kinase 3 ligand (Flt-3L) chimeric cytokine, progenipoietin-1, markedly expands the splenic and hepatic NKT cell population and enhances functional responses to alpha-galactosylceramide. In a murine model of allogeneic stem cell transplantation, donor NKT cells promoted host DC activation and enhanced perforin-restricted CD8+ T cell cytotoxicity against host-type antigens. Following leukemic challenge, donor treatment with progenipoietin-1 significantly improved overall survival when compared with G-CSF or control, attributable to reduced graft-versus-host disease mortality and paradoxical augmentation of graft-versus-leukemia (GVL) effects. Enhanced cellular cytotoxicity was dependent on donor NKT cells, and leukemia clearance was profoundly impaired in recipients of NKT cell-deficient grafts. Enhanced cytotoxicity and GVL effects were not associated with Flt-3L signaling or effects on DCs but were reproduced by prolonged G-CSF receptor engagement with pegylated G-CSF. Thus, modified G-CSF signaling during stem cell mobilization augments NKT cell-dependent CD8+ cytotoxicity, effectively separating graft-versus-host disease and GVL and greatly expanding the potential applicability of allogeneic stem cell transplantation for the therapy of malignant disease.

Induction of natural killer T cell–dependent alloreactivity by administration of granulocyte colony–stimulating factor after bone marrow transplantation

Granulocyte colony–stimulating factor (G-CSF) is often used to hasten neutrophil recovery after allogeneic bone marrow transplantation (BMT), but the clinical and immunological consequences evoked remain unclear. We examined the effect of G-CSF administration after transplantation in mouse models and found that exposure to either standard G-CSF or pegylated-G-CSF soon after BMT substantially increased graft-versus-host disease (GVHD). This effect was dependent on total body irradiation (TBI) rendering host dendritic cells (DCs) responsive to G-CSF by upregulating their expression of the G-CSF receptor. Stimulation of host DCs by G-CSF subsequently unleashed a cascade of events characterized by donor natural killer T cell (NKT cell) activation, interferon-γ secretion and CD40-dependent amplification of donor cytotoxic T lymphocyte function during the effector phase of GVHD. Crucially, the detrimental effects of G-CSF were only present when it was administered after TBI conditioning and at a time when residual host antigen presenting cells were still present, perhaps explaining the conflicting and somewhat controversial clinical studies from the large European and North American BMT registries. These data have major implications for the use of G-CSF in disease states where NKT cell activation may have effects on outcome.

Advances in the understanding of acute graft-versus-host disease

Allogeneic stem cell transplantation (SCT) remains the definitive immunotherapy for malignancy. However, morbidity and mortality due to graft‐vs.‐host disease (GVHD) remains the major barrier to its advancement. Emerging experimental data highlights the immuno‐modulatory roles of diverse cell populations in GVHD, including regulatory T cells, natural killer (NK) cells, NK T cells, γδ T cells, and antigen presenting cells (APC). Knowledge of the pathophysiology of GVHD has driven the investigation of new rational strategies to both prevent severe GVHD and treat steroid‐refractory GVHD. Novel cytokine inhibitors, immune‐suppressant agents known to preserve or even promote regulatory T‐cell function and the depletion of specific alloreactive T‐cell sub‐populations all promise significant advances in the near future. As our knowledge and therapeutic options expand, the ability to limit GVHD whilst preserving anti‐microbial and tumour responses becomes a realistic prospect.

Soluble lymphotoxin is an important effector molecule in GVHD and GVL

Tumor necrosis factor (TNF) is a key cytokine in the effector phase of graft-versus-host disease (GVHD) after bone marrow transplantation, and TNF inhibitors have shown efficacy in clinical and experimental GVHD. TNF signals through the TNF receptors (TNFR), which also bind soluble lymphotoxin (LTalpha3), a TNF family member with a previously unexamined role in GVHD pathogenesis. We have used preclinical models to investigate the role of LT in GVHD. We confirm that grafts deficient in LTalpha have an attenuated capacity to induce GVHD equal to that seen when grafts lack TNF. This is not associated with other defects in cytokine production or T-cell function, suggesting that LTalpha3 exerts its pathogenic activity directly via TNFR signaling. We confirm that donor-derived LTalpha is required for graft-versus-leukemia (GVL) effects, with equal impairment in leukemic clearance seen in recipients of LTalpha- and TNF-deficient grafts. Further impairment in tumor clearance was seen using Tnf/Lta(-/-) donors, suggesting that these molecules play nonredundant roles in GVL. Importantly, donor TNF/LTalpha were only required for GVL where the recipient leukemia was susceptible to apoptosis via p55 TNFR signaling. These data suggest that antagonists neutralizing both TNF and LTalpha3 may be effective for treatment of GVHD, particularly if residual leukemia lacks the p55 TNFR.

Tumor-derived interleukin-4 reduces tumor clearance and deviates the cytokine and granzyme profile of tumor-induced CD8+ T cells

An interleukin (IL)-4-containing tumor environment is reported to be beneficial for immune clearance of tumor cells in vivo; however, the effect of IL-4 on the effector CD8+ T cells contributing to tumor clearance is not well defined. We have used the immunogenic HLA-CW3-expressing P815 (P.CW3) mastocytoma and investigated whether IL-4 expression by the tumor affects tumor clearance and, if so, whether it alters the tumor-induced Vbeta10+ CD8+ T-cell response. P.CW3 were stably transfected with IL-4 or the empty control vector, and independent cell lines were injected i.p. into syngeneic DBA/2 mice. After apparent clearance of primary tumors over 12 to 15 days, secondary tumors arose that lacked surface expression and H-2-restricted antigen presentation of CW3 in part due to the loss of the HLA-CW3 expression cassette. Surprisingly, mice that received IL-4-producing tumor cells showed delayed primary tumor clearance and were significantly more prone to develop secondary tumors compared with mice receiving control tumor cells. Tumor clearance was dependent on CD8+ T cells. The IL-4-secreting P.CW3 tumor cells led to markedly higher mRNA expression of IL-4 and granzyme A and B but no differences in IFN-gamma and IL-2 production, cell proliferation, or ex vivo CTL activity in primary Vbeta10+ CD8+ T cells when compared with the control tumor cells. We concluded that tumor-derived IL-4 selectively changed the quality of the tumor-induced CD8+ T-cell response and resulted in unexpected negative effects on tumor clearance. These data bring into question the delivery of IL-4 to the tumor environment for improving tumor immunotherapy.

Invariant natural killer T cell–natural killer cell interactions dictate transplantation outcome after α-galactosylceramide administration

Invariant natural killer T cells (iNKT cells) have pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects. iNKT cells are activated through their T-cell receptors by glycolipid moieties (typically the alpha-galactosylceramide [alpha-GalCer] derivative KRN7000) presented within CD1d. We investigated the ability of modified alpha-GalCer molecules to differentially modulate alloreactivity and GVL. KRN7000 and the N-acyl variant, C20:2, were administered in multiple well-established murine models of allogeneic stem cell transplantation. The highly potent and specific activation of all type I NKT cells with C20:2 failed to exacerbate and in most settings inhibited GVHD late after transplantation, whereas effects on GVL were variable. In contrast, the administration of KRN7000 induced hyperacute GVHD and early mortality in all models tested. Administration of KRN7000, but not C20:2, was found to result in downstream interleukin (IL)-12 and dendritic cell (DC)-dependent natural killer (NK)- and conventional T-cell activation. Specific depletion of host DCs, IL-12, or donor NK cells prevented this pathogenic response and the induction of hyperacute GVHD. These data demonstrate the ability of profound iNKT activation to modulate both the innate and adaptive immune response via the DC-NK-cell interaction and raise concern for the use of alpha-GalCer therapeutically to modulate GVHD and GVL effects.

The Uhthoff phenomenon : a potential post transplant complication in advanced progressive multiple sclerosis

The Uhthoff phenomenon: a potential post transplant complication in advanced progressive multiple sclerosis

Provision of long-term monitoring and late effects services following adult allogeneic haematopoietic stem cell transplant: a survey of UK NHS-based programmes

Despite international guidelines, optimal delivery models of late effects (LE) services for HSCT patients are unclear from the clinical, organizational and economic viewpoints. To scope current LE service delivery models within the UK NHS (National Health Service), in 2014, we surveyed the 27 adult allogeneic HSCT centres using a 30-question online tool, achieving a 100% response rate. Most LE services were led and delivered by senior physicians (>80% centres). Follow-up was usually provided in a dedicated allograft or LE clinic for the first year (>90% centres), but thereafter attrition meant that only ~50% of patients were followed after 5 years. Most centres (69%) had a standard operating procedure for long-term monitoring but access to a LE Multi-Disciplinary Team was rare (19% centres). Access to medical specialities necessary for LE management was good, but specialist interest in long-term HSCT complications was uncommon. Some screening (endocrinopathy, cardiovascular) was near universal, but other areas were more limited (mammography, cervical smears). Funding of extra staff and investigations were the most commonly perceived barriers to implementation of LE services. This survey shows variation in the long-term follow-up of allogeneic HSCT survivors within the UK NHS and further work is warranted to optimize effective, sustainable and affordable models of LE service delivery among this group.

Donor treatment with a multipegylated G-CSF maximizes graft-versus-leukemia effects.

Donor treatment with granulocyle-colony stimulating factor (G-CSF) is known to modulate immune function, characterized by the generation of regulatory myelogenous and T cell populations and Th2 differentiation. Recently, these effects have been shown to be enhanced by pegylation of the G-CSF molecule, which also improves graft-versus-leukemia (GVL) via activation of invariant natural killer (iNK) T cells. We have compared G-CSF bound to a single PEG molecule (monopeg-G-CSF) as used clinically to a G-CSF molecule bound to multiple PEG molecules (multipeg-G-CSF) in major histocompatibility complex (MHC) disparate and matched models of graft-versus-host disease (GVHD) and GVL. We demonstrate that multipeg-G-CSF induces greater levels of progenitor cell, myelogenous, and iNKT cell expansion than monopeg-G-CSF, while inducing similar protection from GVHD. Despite this, multipeg-G-CSF enhanced CTL function in vivo and improved iNKT cell-dependent leukemia clearance. Thus, GVL and GVHD can be further separated after allogeneic stem cell transplantation by mobilization with a multiple-pegylated G-CSF molecule.