Antonio Pérez-Martínez

Comparison of primary human cytotoxic T-cell and natural killer cell responses reveal similar molecular requirements for lytic granule exocytosis but differences in cytokine production.

Cytotoxic lymphocytes, encompassing cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, kill pathogen-infected, neoplastic, or certain hematopoietic cells through the release of perforin-containing lytic granules. In the present study, we first performed probability-state modeling of differentiation and lytic granule markers on CD8(+) T cells to enable the comparison of bona fide CTLs with NK cells. Analysis identified CD57(bright) expression as a reliable phenotype of granule marker-containing CTLs. We then compared CD3(+)CD8(+)CD57(bright) CTLs with NK cells. Healthy adult peripheral blood CD3(+)CD8(+)CD57(bright) CTLs expressed more granzyme B but less perforin than CD3(-)CD56(dim) NK cells. On stimulation, such CTLs degranulated more readily than other T-cell subsets, but had a propensity to degranulate that was similar to NK cells. Remarkably, the CTLs produced cytokines more rapidly and with greater frequency than NK cells. In patients with biallelic mutations in UNC13D, STX11, or STXBP2 associated with familial hemophagocytic lymphohistiocytosis, CTL and NK cell degranulation were similarly impaired. Therefore, cytotoxic lymphocyte subsets have similar requirements for Munc13-4, syntaxin-11, and Munc18-2 in lytic granule exocytosis. The present results provide a detailed comparison of human CD3(+)CD8(+)CD57(bright) CTLs and NK cells and suggest that analysis of CD57(bright) CTL function may prove useful in the diagnosis of primary immunodeficiencies including familial hemophagocytic lymphohistiocytosis.

High-dose chemotherapy with autologous stem cell rescue for children with high risk and recurrent medulloblastoma and supratentorial primitive neuroectodermal tumors

Current treatment for high risk and recurrent medulloblastoma (MB) and supratentorial primitive neuroectodermal tumors (stPNET) has a very poor prognosis in children. High dose chemotherapy (HDCT) and autologous stem cell rescue have improved survival rates. We present 19 patients (thirteen classified in the high risk group and six patients with recurrent disease) that received HDCT and autologous stem cell rescue.In the high risk group [Med Pediatr Oncol 38 (2002) 83], all patients underwent neurosurgical debulking. Standard chemotherapy was prescribed in 10 patients. Radiotherapy was given to 4 patients (all older than 4 years old). In the recurrence disease group [Childs Nerv Syst 15 (1999) 498], five patients underwent surgery. Radiotherapy was given to those who were not previously irradiated. The HDCT in twelve patients consisted of busulfan 4 mg/kg/day, orally over 4 days in 6-hourly divided doses and melphalan at a dose of 140 mg/m2/day by intravenous infusion over 5 min on day −1. Three patients additionally received thiotepa 250 mg/m2/day intravenously over 2 days and four patients additionally received topotecan 2 mg/m2/day over 5 days by intravenous infusion over 30 min. The other seven patients received busulfan and thiotepa at the same doses.Patient’s stem cells were mobilized with granulocyte colony-stimulating factor at a dose of 12 μg/kg twice daily subcutaneously for four consecutive days. Cryopreserved peripheral blood progenitor cells were re-infused 48 h after completion of chemotherapy. With a median follow-up of 34 months (range 5–93) eight complete responses and one partial response were observed. Three patients died of treatment-related toxicities (15%). The 2 year event-free survival was 37.67 ± 14% in all patients and 57 ± 15% for the high risk group.Therefore we conclude that HDCT may improve survival rates in patients with high risk/recurrent MB and stPNET despite treatment toxicity.

Mobilisation of mesenchymal cells into blood in response to skeletal muscle injury

Mesenchymal cells recruited to damaged tissues must circulate through the bloodstream. The absolute numbers of circulating mesenchymal stem cells (cMSCs) in two different models of acute and chronic skeletal muscle injury were determined. cMSCs were present in significantly higher numbers in both models than in healthy controls. These results support the hypothesis that MSCs are mobilised into the bloodstream after skeletal muscle tissue damage. These two models (acute and chronic) would be of value in the search for molecular mediators of mobilisation of MSCs into the circulation.

KIR-HLA receptor-ligand mismatch associated with a graft-versus-tumor effect in haploidentical stem cell transplantation for pediatric metastatic solid tumors.

Killer immunoglobulin‐like receptors (KIRs) on natural killer cells (NKs) recognize groups of human leukocyte antigen (HLA) class I alleles. Cells without an inhibitory HLA ligand may trigger NK activation. Reduced risk of relapse has been reported in malignant hematologic diseases after haploidentical transplantation when HLA ligands against the inhibitory KIRs present in the donor were absent in the recipient. We performed haploidentical transplant in three children with refractory solid tumors. Our results showed that beneficial antitumor effects could be observed in the presence of inhibitory KIR–HLA mismatch. These preliminary results suggest a possible association between disease control and NK cell alloreactivity. Pediatr Blood Cancer 2009;53:120–124.

A phase I/II trial of interleukin-15--stimulated natural killer cell infusion after haplo-identical stem cell transplantation for pediatric refractory solid tumors.

BACKGROUND AIMS Preliminary data suggest that T-cell-depleted haplo-identical stem cell transplantation (haplo-SCT) has a clinically beneficial allograft-versus-tumor effect associated with natural killer (NK) cell immune reconstitution. METHODS This phase I/II trial descriptively evaluates the feasibility of interleukin (IL)-15-stimulated NK cell infusion after haplo-SCT in pediatric patients with refractory solid tumors. RESULTS Six patients received an IL-15-stimulated NK cell infusion at 30 days after haplo-SCT. The mean number of infused NK cells per product was 11.3 × 10(6)/kg (range, 3-27 × 10(6)/kg). The T-cell count was <1 × 10(3)/kg in all patients (range, 0-0.75 × 10(3)/kg). No toxic effects related to IL-15--stimulated NK cell infusion were observed. Four of the six patients showed a clinical response (one achieved very good partial remission, two achieved partial remission and one had stable disease). One patient had progressive disease, and the response was not evaluated in the remaining patient. After a median follow-up period of 310 days, all patients had died: four of cancer relapse, one of cancer-associated thrombotic micro-angiopathy and one of acute graft-versus-host disease. CONCLUSIONS The adoptive transfer of allogeneic IL-15--stimulated NK cells might be feasible and safe in heavily pretreated pediatric patients with refractory solid tumors, though the advanced stage of disease and toxic effects of haplo-SCT may limit the efficacy of NK cell infusion in this population.

Peripheral blood progenitor cell collection adverse events for childhood allogeneic donors: variables related to the collection and safety profile.

The use of children as haematopoietic stem cell donors represents an ethical dilemma. For some investigators, this is even more questionable if the children underwent peripheral blood progenitor cell (PBPC) collection. Due to these controversies, information related to adverse events (AE) in paediatric donors and variables related to the collection is scanty. We analysed 152 PBPC collections, and compared the complications and results between young children, older children and adults. The pattern of AE was shown to vary according to the age of the donor. Older paediatric donors and adults had a higher incidence of complaints related to PBPC priming (54·3% vs. 79·7%, respectively) than the youngest children (12%). On the other hand, these donors had a lower incidence of AE during PBPC collection (19·6% older children, 37·3% adults) mainly related to hypocalcaemia, than the youngest donors, who suffered mainly cardiovascular complications due to hypovolaemia (51·7%). The only variables related to collected cell dose were total blood volume processed per donor body weight, and CD34+ cell count before apheresis. The donor/recipient body weight ratio predicted the outcome of collection in a single large volume leukapheresis. Donors with body weight ratio ≥0·75 had 4·69 times higher likelihood to reach the minimum target cell dose.

Natural killer cells can exert a graft-vs-tumor effect in haploidentical stem cell transplantation for pediatric solid tumors

Little progress has been made with regard to the survival of children with metastatic and refractory solid tumors. Preliminary data from haploidentical stem cell transplantation (haplo-SCT) suggested a clinically beneficial allograft-vs-tumor effect associated with natural killer cell (NK) donor-recipient mismatch. We hypothesized that interaction between activatory receptors on NK cells and their ligands on tumor cells could be also important. To evaluate the NK-cell-mediated allograft-vs-tumor effect, we conducted a pilot study of haplo-SCT on six children with refractory solid tumors. Our specific goal for this study was NKG2D-major histocompatibility complex class I-related chain A interaction. Tasks include specific immunoassays that support haplo-SCT in refractory solid tumors. Patients suffered from neuroblastoma (n = 1), Ewing sarcoma (n = 2), a desmoplastic tumor (n = 1), nasopharyngeal carcinoma (n = 1), and embryonal rhabdomyosarcoma (n = 1). Pretransplantation disease status showed progressive disease in 2 patients, partial remission in 2 patients, and complete remission in 2 patients. NK-cell mismatch was present in three donor-recipients. Ligands for NKG2D receptors, major histocompatibility complex class I-related chain A and UL16 binding protein 2 were overexpressed in six of six and four of six tumors, respectively. NK cells led early immune reconstitution. After haplo-SCT, three patients were in complete remission, one patient showed partial remission, and two patients were in stable disease. With a median follow-up of 14 months, three patients were alive and in complete remission, and three patients had died; two due to progressive disease and one of transplant-related toxicity. Blocking NKG2D-major histocompatibility complex class I-related chain A interaction in vitro reduced NK-cell cytotoxicity. Our preliminary results suggest a beneficial effect from haplo-SCT in refractory solid tumors.

Early evaluation of immune reconstitution following allogeneic CD3/CD19-depleted grafts from alternative donors in childhood acute leukemia

Graft engineering procedures for hematopoietic SCT (HSCT) may improve the chance of success in matched unrelated donor (MUD) and haploidentical donor transplantations. Successful donor immune reconstitution is important to mediate GVL effects in reduced-intensity conditioning (RIC) HSCT. We prospectively investigated early immune reconstitution and clinical outcome in 30 CD3/CD19-depleted MUD (n=15) or HP (n=15) HSCTs for high-risk childhood leukemia using a fludarabine-based RIC without serotherapy. The graft consisted of a mean of 10.5 × 106/kg CD34+, 77 × 103/kg CD3+ and 39 × 106/kg CD56+ cells. After transplantation, 86% of the patients engrafted. In all, 13% of patients had >grade 3 acute GVHD. Natural killer (NK) cell, DC and T-cell recovery achieved normal values within the first 60 days after transplantation. DC recovery was dominated by the DC2− subset. NK-cell phenotype was altered and cytotoxicity was lower compared with their donors. EFS was 50±9% (73±11% for those in CR1 and 26±11% for those with advanced disease). Faster DC2− recovery was associated with better outcome, especially in the MUD setting. In summary, CD3/CD19-depleted HSCT with fludarabine-based RIC without serotherapy resulted in favorable patient survival, and rapid NK, DC and T-cell recovery.

Blood dendritic cells suppress NK cell function and increase the risk of leukemia relapse after hematopoietic cell transplantation

NK cells play an important role in hematopoietic stem cell transplantation (HCT) and in cross talk with dendritic cells (DCs) to induce primary T cell response against infection. Therefore, we hypothesized that blood DCs should augment NK cell function and reduce the risk of leukemia relapse after HCT. To test this hypothesis, we conducted laboratory and clinical studies in parallel. We found that although, phenotypically, NK cells could induce DC maturation and DCs could in turn increase activating marker expression on NK cells, paradoxically, both BDCA1(+) myeloid DCs and BDCA4(+) plasmacytoid DCs suppressed the function of NK cells. Patients who received an HLA-haploidentical graft containing a larger number of BDCA1(+) DCs or BDCA4(+) DCs had a higher risk of leukemia relapse and poorer survival. Further experiments indicated that the potent inhibition on NK cell cytokine production and cytotoxicity was mediated in part through the secretion of IL-10 by BDCA1(+) DCs and IL-6 by BDCA4(+) DCs. These results have significant implications for future HCT strategies.

In vitro Natural Killer Cell Immunotherapy for Medulloblastoma.

How the immune system attacks medulloblastoma (MB) tumors effectively is unclear, although natural killer (NK) cells play an important role in immune defense against tumor cells. Interactions between receptors on NK cells and ligands expressed by tumor cells are critical for tumor control by immunotherapy. In this study, we analyzed tumor samples from 54 MB patients for expression of major histocompatibility complex class I-related chains A (MICA) and UL16 binding protein (ULPB-2), which are ligands for the NK group 2 member D activatory receptor (NKG2D). The percentage of MICA and ULBP-2 positive cells was higher than 25% in 68% and 6% of MB patients, respectively. A moderate-high intensity of MICA cytoplasmic staining was observed in 46% MB patients and weak ULBP-2 staining was observed in 8% MB patients. No correlation between MICA/ULBP-2 expression and patient outcome was found. We observed that HTB-186, a MB cell line, was moderately resistant to NK cell cytotoxicity in vitro. Blocking MICA/ULBP-2 on HTB-186, and NKG2D receptor on NK cells increased resistance to NK cell lysis in vitro. However, HLA class I blocking on HTB-186 and overnight incubation with IL-15 stimulated NK cells efficiently killed tumor cells in vitro. We conclude that although NKG2D/MICA-ULBP-2 interactions have a role in NK cell cytotoxicity against MB, high expression of HLA class I can protect MB from NK cell cytotoxicity. Even so, our in vitro data indicate that if NK cells are appropriately stimulated, they may have the potential to target MB in vivo.