RESUMEN
How hematopoietic stem and progenitor cell (HSPC) fate decisions are affected by genetic alterations acquired during AML leukemogenesis is poorly understood and mainly explored in animal models. Here, we study isocitrate dehydrogenase (IDH) gene mutations in the human model of HSPC and discuss the available literature on this topic. IDH1/2 mutations occur in ~20% of AML cases, are recognized among the mutations earliest acquired during leukemogenesis, and are targets of specific inhibitors (ivosidenib and enasidenib, respectively). In order to investigate the direct effects of these mutations on HSPCs, we expressed IDH1-R132H or IDH2-R140Q mutants into human CD34+ healthy donor cells via lentiviral transduction and analyzed the colony-forming unit (CFU) ability. CFU ability was dramatically compromised with a complete trilineage block of differentiation. Strikingly, the block was reversed by specific inhibitors, confirming that it was a specific effect induced by the mutants. In line with this observation, the CD34+ leukemic precursors isolated from a patient with IDH2-mutated AML at baseline and during enasidenib treatment showed progressive and marked improvements in their fitness over time, in terms of CFU ability and propensity to differentiate. They attained clonal trilinear reconstitution of hematopoiesis and complete hematological remission.
RESUMEN
Blinatumomab alone or with donor leukocyte infusions (DLI) has been used after allogeneic hematopoietic stem cell transplantation (HSCT) as a salvage therapy in relapsing patients with CD19+ hematological malignancies. It was effective in a fraction of them, with low incidence of Graft-versus-Host Disease (GvHD). Immunosuppressive drugs used as GvHD prophylaxis hinder T cell function and reduce the efficacy of the treatment. Because T cell-depleted haploidentical HSCT with donor regulatory and conventional T cells (Treg/Tcon haploidentical HSCT) does not require post-transplant immunosuppression, it is an ideal platform for the concomitant use of blinatumomab and DLI. However, the risk of GvHD is high because the donor is haploidentical. We treated two patients with CD19+ acute lymphoblastic leukemia (ALL) who had relapsed after Treg/Tcon haploidentical HSCT with blinatumomab and DLI. Despite the mismatch for one HLA haplotype, they did not develop GvHD and achieved complete remission with negative minimal residual disease. Consistently, we found that blinatumomab did not enhance T cell alloreactivity in vitro. Eventually, the two patients relapsed again because of their high disease risk. This study suggests that treatment with blinatumomab and DLI can be feasible to treat relapse after haploidentical transplantation, and its pre-emptive use should be considered to improve efficacy.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Linfocitos T/patologíaRESUMEN
Allogeneic hematopoietic stem cell transplantation (HSCT) is the most effective treatment in eradicating high-risk acute myeloid leukemia (AML). Here, we present data from a novel HLA-haploidentical HSCT protocol that addressed the 2 remaining major unmet medical needs: leukemia relapse and chronic graft-versus-host disease (cGVHD). Fifty AML patients were enrolled in the study. The conditioning regimen included total body irradiation for patients up to age 50 years and total marrow/lymphoid irradiation for patients age 51 to 65 years. Irradiation was followed by thiotepa, fludarabine, and cyclophosphamide. Patients received an infusion of 2 × 106/kg donor regulatory T cells on day -4 followed by 1 × 106/kg donor conventional T cells on day -1 and a mean of 10.7 × 106 ± 3.4 × 106/kgpurified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GVHD prophylaxis was administered posttransplantation. Patients achieved full donor-type engraftment. Fifteen patients developed grade ≥2 acute GVHD (aGVHD). Twelve of the 15 patients with aGVHD were alive and no longer receiving immunosuppressive therapy. Moderate/severe cGVHD occurred in only 1 patient. Nonrelapse mortality occurred in 10 patients. Only 2 patients relapsed. Consequently, at a median follow-up of 29 months, the probability of moderate/severe cGVHD/relapse-free survival was 75% (95% confidence interval, 71%-78%). A novel HLA-haploidentical HSCT strategy that combines an age-adapted myeloablative conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy resulted in an unprecedented cGVHD/relapse-free survival rate in 50 AML patients with a median age of 53 years. This trial was registered with the Umbria Region Institutional Review Board Public Registry as identification code 02/14 and public registry #2384/14 and at www.clinicaltrials.gov as #NCT03977103.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Anciano , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Leucemia Mieloide Aguda/terapia , Persona de Mediana Edad , Acondicionamiento Pretrasplante , Irradiación Corporal TotalRESUMEN
Dysregulated NOTCH1 signaling, by either gene mutations or microenvironment interactions, has been increasingly linked to chronic lymphocytic leukemia (CLL). Thus, inhibiting NOTCH1 activity represents a potential therapeutic opportunity for this disease. Using gene expression-based screening, we identified the calcium channel modulator bepridil as a new NOTCH1 pathway inhibitor. In primary CLL cells, bepridil induced selective apoptosis even in the presence of the protective stroma. Cytotoxic effects of bepridil were independent of NOTCH1 mutation and other prognostic markers. The antitumor efficacy of bepridil was associated with inhibition of NOTCH1 activity through a decrement in trans-membrane and activated NOTCH1 protein levels with unchanged NOTCH2 protein levels. In a CLL xenotransplant model, bepridil significantly reduced the percentage of leukemic cells infiltrating the spleen via enhanced apoptosis and decreased NOTCH1 activation. In conclusion, we report in vitro and in vivo anti-leukemic activity of bepridil associated with inhibition of the NOTCH1 pathway in CLL. These data provide a rationale for the clinical development of bepridil as anti-NOTCH1 targeted therapy for CLL patients.
Asunto(s)
Antineoplásicos/farmacología , Bepridil/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/metabolismo , Animales , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Quimiotaxis/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Mutación , Pronóstico , Receptor Notch1/genética , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We developed a good manufacturing practices-compatible expansion protocol to improve number and purity of regulatory T cells (Tregs) available for clinical trials. Six clinical-grade separation procedures were performed, followed by expansion with high-dose interleukin (IL)-2, anti-CD3/anti-CD28 TCR stimulation, and rapamycin for 19 days achieving a median of 8.5-fold (range, 6.25 to 13.7) expansion. FOXP3 expression was stably maintained over the culture period, while the percentage of CD127 was significantly reduced. The in vitro suppression assay showed a strong Mixed Lymphocytes Reaction inhibition. In vitro amplification did not induce any karyotypic modification. To evaluate the graft-versus-host disease (GVHD)/graft-versus-leukemia (GVL) bifunctional axis, expanded Tregs and conventional T cells (Tcons) were tested in NOD/SCID/IL2Rgnull mice injected with primary acute myeloid leukemia (AML) cells, AML cell line, acute lymphoid leukemia Philadelphia cell line, or Burkitt-like lymphoma cell line. All mice that received leukemia cells together with expanded Tregs and Tcons were rescued from leukemia and survived without GVHD, showing that Treg expansion procedure did not compromise GVHD control and the strong Tcon-mediated GVL activity. This report might represent the basis for treating high-risk leukemia and/or relapsed/refractory leukemia patients with high-dose Treg/Tcons.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Linfocitos T Reguladores/metabolismo , Trasplante Haploidéntico/métodos , Animales , Modelos Animales de Enfermedad , Efecto Injerto vs Leucemia , Ratones , Ratones Endogámicos NOD , Ratones SCIDRESUMEN
Posttransplant relapse is still the major cause of treatment failure in high-risk acute leukemia. Attempts to manipulate alloreactive T cells to spare normal cells while killing leukemic cells have been unsuccessful. In HLA-haploidentical transplantation, we reported that donor-derived T regulatory cells (Tregs), coinfused with conventional T cells (Tcons), protected recipients against graft-versus-host disease (GVHD). The present phase 2 study investigated whether Treg-Tcon adoptive immunotherapy prevents posttransplant leukemia relapse. Forty-three adults with high-risk acute leukemia (acute myeloid leukemia 33; acute lymphoblastic leukemia 10) were conditioned with a total body irradiation-based regimen. Grafts included CD34(+) cells (mean 9.7 × 10(6)/kg), Tregs (mean 2.5 × 10(6)/kg), and Tcons (mean 1.1 × 10(6)/kg). No posttransplant immunosuppression was given. Ninety-five percent of patients achieved full-donor type engraftment and 15% developed ≥grade 2 acute GVHD. The probability of disease-free survival was 0.56 at a median follow-up of 46 months. The very low cumulative incidence of relapse (0.05) was significantly better than in historical controls. These results demonstrate the immunosuppressive potential of Tregs can be used to suppress GVHD without loss of the benefits of graft-versus-leukemia (GVL) activity. Humanized murine models provided insights into the mechanisms underlying separation of GVL from GVHD, suggesting the GVL effect is due to largely unopposed Tcon alloantigen recognition in bone marrow.
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Trasplante de Médula Ósea , Efecto Injerto vs Leucemia/inmunología , Inmunoterapia Adoptiva , Leucemia Mieloide Aguda/terapia , Recurrencia Local de Neoplasia/prevención & control , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfocitos T Reguladores/inmunología , Adolescente , Adulto , Anciano , Animales , Modelos Animales de Enfermedad , Femenino , Estudios de Seguimiento , Antígenos HLA/inmunología , Histocompatibilidad , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/mortalidad , Depleción Linfocítica , Masculino , Ratones , Persona de Mediana Edad , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Pronóstico , Inducción de Remisión , Tasa de Supervivencia , Acondicionamiento Pretrasplante , Trasplante Homólogo , Adulto JovenRESUMEN
We selected T regulatory cells (Tregs) from standard leukapheresis using double-negative selection (anti-CD8 and anti-CD19) followed by positive selection (anti-CD25) and 72 procedures were performed. A median of 263×10(6)cells (range 143-470×10(6)) were recovered with a mean of CD4(+)/CD25(+) cells of 94.5±2.4% (36.5±18.6% CD4(+)/CD25(+hi)). FoxP3(+) cells were equal to 79.8%±22.2. CD127(+) cells were 12.5%±8.2. The inhibition assay showed an inhibition rate of 67±22. Cells isolated by means of this approach can be used in allogeneic hematopoietic stem cell transplantation to reduce the incidence and severity of GvHD without bystander inhibition of general immunity.
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Linfocitos T Reguladores/citología , Animales , Separación Celular/métodos , Modelos Animales de Enfermedad , Humanos , Inmunofenotipificación , Leucaféresis , Ratones , Linfocitos T Reguladores/inmunologíaRESUMEN
Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)-haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when co-infused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.
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Enfermedad Injerto contra Huésped/prevención & control , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas , Histocompatibilidad/inmunología , Sistema Inmunológico/fisiología , Linfocitos T Reguladores/fisiología , Adulto , Femenino , Enfermedad Injerto contra Huésped/inmunología , Neoplasias Hematológicas/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Prueba de Histocompatibilidad , Humanos , Sistema Inmunológico/inmunología , Masculino , Persona de Mediana Edad , Recurrencia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Acondicionamiento Pretrasplante/métodos , Inmunología del Trasplante/fisiología , Trasplante Homólogo , Adulto JovenRESUMEN
Much progress has been made in the clinical, biological and technical aspects of the T-cell-depleted full-haplotype mismatched transplants for acute leukemia. Our experience demonstrates that infusing a megadose of extensively T-cell-depleted hematopoietic peripheral blood stem cells after an immuno-myeloablative conditioning regimen in acute leukemia patients ensures sustained engraftment with minimal graft-vs-host disease (GvHD) without the need of any post-transplant immunosuppressive treatment. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in more than 200 high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.
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Haplotipos , Trasplante de Células Madre Hematopoyéticas , Leucemia/terapia , Donantes de Tejidos , Adolescente , Adulto , Niño , Preescolar , Supervivencia sin Enfermedad , Enfermedad Injerto contra Huésped , Humanos , Incidencia , Leucemia/epidemiología , Leucemia/mortalidad , Persona de Mediana Edad , Recurrencia , Resultado del TratamientoRESUMEN
T-cell homeostasis is regulated by several molecules; among these, interleukin (IL)-7 plays an essential role in the survival and homeostatic proliferation of peripheral naive T cells. In a previous study, we investigated whether human mesenchymal stromal cells (MSCs) could be engineered with the IL-7 gene to produce functional level of this cytokine. In the present study, we analyzed the impact of different quantities of IL-7 produced by MSCs on the survival and proliferation of a negative immunoselected naive (CD3(+)/CD45RA(+)) T-cell population. Co-cultivation of peripheral naive T cells with MSCs producing low (16 pg/mL) or high (1000 pg/mL) IL-7 levels or in the presence of exogenous IL-7 (0.01 ng/mL and 100 ng/mL) maintained the CD3(+)/CD45RA(+) naive T-cell phenotype. Chemokine receptor CCR7(+) expression was also maintained among this T-cell population. Naive T-cell molecular characteristics were maintained as assessed by the Vbeta spectratyping complexity score, which showed the maintenance of a broad T-cell repertoire. No Th1 or Th2 differentiation was observed, as assessed by interferon-gamma or IL-4 accumulation. In contrast, only MSCs producing high amounts of IL-7 caused increased activation (CD25 31.2% +/- 12% vs 10% +/- 3.5%; P < .05), proliferation (CD71 17.8+/-7% vs 9.3%+/-3, P < .05), apoptosis (assessed by annexin V: 18.6% +/- 5% vs 14.9% +/- 2.6%; P > .05), and the phase S cell cycle (15% vs 6.9%, P > .05). Exogenous IL-7 exhibited no significant effect. In conclusion, we demonstrated that IL-7 produced by MSCs has a dose-independent effect on naive T-cell survival while exerting a dose-dependent effect on activation/proliferation. Due to the continuous production of IL-7 by engineered cells, our system is more efficacious than exogenous IL-7.
Asunto(s)
Interleucina-7/fisiología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/efectos de los fármacos , Subgrupos de Linfocitos T/citología , Antígenos CD/análisis , Apoptosis , Células Cultivadas/citología , Células Cultivadas/metabolismo , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Ingeniería Genética , Vectores Genéticos/genética , Humanos , Inmunofenotipificación , Interleucina-7/genética , Interleucina-7/metabolismo , Interleucina-7/farmacología , Células Madre Mesenquimatosas/metabolismo , Receptores CCR7 , Receptores de Quimiocina/análisis , Proteínas Recombinantes de Fusión/fisiología , Retroviridae/genética , Fase S , Subgrupos de Linfocitos T/inmunología , Transducción GenéticaRESUMEN
In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%). IL-7 production was 16.37 (+/-2 SD) pg/ml, which remained stable for 60 days. In vitro-immunoselected naive T cells maintained the CD45RA+ CD45RO- naive phenotype (4.2 times more than controls) after 7 days of culture with IL-7-engineered stromal cells. The apoptosis rate (4.7%) of the naive T cells cultured with transduced stromal cells overlapped with that of freshly isolated cells. Immunohistological analysis detected stromal cells in bone marrow, spleen, and thymus. Cotransplantation of IL-7-engineered stromal cells with CD34+ cells improved engraftment in terms of CD45+ cells and significantly increased the CD3+ cell count in peripheral blood, bone marrow, and spleen. These data demonstrate the following: (1) human stromal cells can be transduced, generating a normal layer; (2) transduced stromal cells in vitro maintain the naive T cell phenotype; and (3) IL-7-transduced stromal cells in vivo home to lymphoid organs and produce sufficient IL-7 in loco, supporting T cell development in a cotransplantation model. Because of their efficient cytokine production and homing, IL-7-engineered stromal cells might be an ideal vehicle to hasten immunological reconstitution in T cell-depleted hosts.
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Ingeniería Genética/métodos , Interleucina-7/genética , Células del Estroma/fisiología , Linfocitos T/inmunología , Animales , Antígenos CD34/inmunología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Trasplante de Células/métodos , Humanos , Interleucina-7/inmunología , Interleucina-7/metabolismo , Antígenos Comunes de Leucocito/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Retroviridae/genética , Linfocitos T/fisiología , Transducción GenéticaRESUMEN
Haploidentical stem cell transplantation has became a clinical reality in the last 10 years as it provides the chance of transplant for about 50% of patients with hematological malignancies who do not have a matched related or unrelated donor. Proper graft preparation for this type of transplant is crucial and this paper analyses our work over the past decade in the search for the optimal graft processing procedure moving from E-rosetting and soybean agglutination, through a combination of negative or positive selection of hematopoietic stem cells to the current method of one-step positive selection. In preparing a graft for haploidentical transplant, three essential requisites must be met. It must contain (1) a megadose (>10 x 10(6) x kg recipient b.w.) of hematopoietic stem cells to overcome the HLA histocompatibility barrier; (2) very few T-lymphocytes (CD3+ cells < 3 x 10(4)/kg recipient b.w.) to prevent severe acute and chronic graft-versus-host disease (GvHD); (3) very few B-lymphocytes to prevent Epstein-Barr virus-related lymphoproliferative disorders. With current graft processing technologies based on positive selection of hematopoietic stem cells, these requirements can be met. A 70-80% hematopoietic stem cell recovery ensures the target megadose is achieved in over 70% of cases with a T-cell depletion of more than 4 logs and a B-cell depletion of over 3 logs. Progress in graft processing has ensured primary, sustained engraftment rates of over 90% and has significantly reduced the incidence of severe acute GvHD and EBV-related lymphoproliferative disorders. Modern time-saving automated graft processing devices ensure reproducibility, reliability, and biological safety, which make widespread application of the haploidentical transplant currently feasible.