Tatjana Banovic

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.

Conventional dendritic cells are the critical donor APC presenting alloantigen after experimental bone marrow transplantation

We have quantified the relative contribution of donor antigen-presenting cell populations to alloantigen presentation after bone marrow transplantation (BMT) by using transgenic T cells that can respond to host-derived alloantigen presented within the donor major histocompatibility complex. We also used additional transgenic/knockout donor mice and/or monoclonal antibodies that allowed conditional depletion of conventional dendritic cells (cDCs), plasmacytoid DC (pDCs), macrophages, or B cells. Using these systems, we demonstrate that donor cDCs are the critical population presenting alloantigen after BMT, whereas pDCs and macrophages do not make a significant contribution in isolation. In addition, alloantigen presentation was significantly enhanced in the absence of donor B cells, confirming a regulatory role for these cells early after transplantation. These data have major implications for the design of therapeutic strategies post-BMT, and suggest that cDC depletion and the promotion of B-cell reconstitution may be beneficial tools for the control of alloreactivity.

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.

Disseminated Varicella Infection Caused by Varicella Vaccine Strain in a Child With Low Invariant Natural Killer T Cells and Diminished CD1d Expression

BACKGROUND Live attenuated varicella vaccine is considered a safe vaccine with serious adverse effects reported only in immunocompromised children. We describe a severe life-threatening infection with varicella vaccine virus causing rash and pneumonitis in a 6-year-old boy with no apparent immunodeficiency. METHODS AND RESULTS Polymerase chain reaction (PCR) analysis of vesicle swab samples demonstrated varicella zoster virus (VZV). Sequencing of the PCR product demonstrated 100% homology with human herpesvirus 3 strain VZV-Oka ORF62 gene. Routine immunologic investigations failed to demonstrate any abnormality. Total leukocyte, lymphocyte, and neutrophil counts and lymphocyte subsets were normal. Immunoglobulins, C3, C4, and CH50 were intact. Specific IgG to protein and polysaccharide antigens and to Epstein-Barr virus and cytomegalovirus were present. Normal lymphocyte proliferation to phytohemagglutinin and VZV antigens was detected. Neutrophil function and natural killer (NK) cell activity were normal. The analysis of invariant NK T (iNKT) cell numbers and function revealed diminished iNKT cells, reported once previously and unique to our patient, deficient expression of the cognate receptor, CD1d. CONCLUSIONS This report provides a further link between deficiency of the iNKT/CD1d pathway and increased susceptibility to varicella vaccine virus, suggesting an important role of this innate pathway in host defense against yet another member of the herpesvirus family.

Graft-versus-Host Disease Prevents the Maturation of Plasmacytoid Dendritic Cells

The role of Ag presenting cell subsets in graft-versus-host disease (GVHD) remains unclear. We have thus examined the ability of plasmacytoid dendritic cells (pDC) to modulate transplant outcome. Surprisingly, host pDC were exquisitely sensitive to total body irradiation and were depleted before transplantation, thus allowing us to focus on donor pDC. The depletion of all pDC from bone marrow grafts resulted in an acceleration of GVHD mortality while the depletion of mature pDC from G-CSF mobilized splenic grafts had no effect. Thus, donor bone marrow pDC, but not mature pDC contained within stem cell grafts attenuate acute GVHD. In the presence of GVHD, donor pDC completely failed to reconstitute although a CD11clow120G8+ precursor DC reconstituted in an exaggerated and transient manner. These cells expressed Flt-3, the macrophage colony stimulating factor receptor and, consistent with a common dendritic cell (DC) precursor, were capable of differentiation into pDC and conventional DC in vivo in the absence of GVHD. These precursors were MHC class II+ and CD80/86+ but lacked CD40, were actively presenting host Ag and inhibited GVHD and T cell proliferation in a contact-dependent fashion. These data demonstrate that GVHD prevents the maturation of pDC and instead promotes the generation of a suppressive precursor DC, further contributing to the state of immune paralysis after transplantation.

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.

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.

A critical role for donor‐derived IL‐22 in cutaneous chronic GVHD

Graft‐versus‐host disease (GVHD) is the major cause of nonrelapse morbidity and mortality after allogeneic stem cell transplantation (allo‐SCT). Prevention and treatment of GVHD remain inadequate and commonly lead to end‐organ dysfunction and opportunistic infection. The role of interleukin (IL)‐17 and IL‐22 in GVHD remains uncertain, due to an apparent lack of lineage fidelity and variable and contextually determined protective and pathogenic effects. We demonstrate that donor T cell–derived IL‐22 significantly exacerbates cutaneous chronic GVHD and that IL‐22 is produced by highly inflammatory donor CD4+ T cells posttransplantation. IL‐22 and IL‐17A derive from both independent and overlapping lineages, defined as T helper (Th)22 and IL‐22+ Th17 cells. Donor Th22 and IL‐22+ Th17 cells share a similar IL‐6–dependent developmental pathway, and while Th22 cells arise independently of the IL‐22+Th17 lineage, IL‐17 signaling to donor Th22 directly promotes their development in allo‐SCT. Importantly, while both IL‐22 and IL‐17 mediate skin GVHD, Th17‐induced chronic GVHD can be attenuated by IL‐22 inhibition in preclinical systems. In the clinic, high levels of both IL‐17A and IL‐22 expression are present in the skin of patients with GVHD after allo‐SCT. Together, these data demonstrate a key role for donor‐derived IL‐22 in patients with chronic skin GVHD and confirm parallel but symbiotic developmental pathways of Th22 and Th17 differentiation.

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.