RESUMO
Tumor-associated antigens (TAAs) are monomorphic self-antigens that are proposed as targets for immunotherapeutic approaches to treat malignancies. We investigated whether T cells with sufficient avidity to recognize naturally overexpressed self-antigens in the context of self-HLA can be found in the T-cell repertoire of healthy donors. Minor histocompatibility antigen (MiHA)-specific T cells were used as a model, as the influence of thymic selection on the T-cell repertoire directed against MiHA can be studied in both self (MiHApos donors) and non-self (MiHAneg donors) backgrounds. T-cell clones directed against the HLA*02:01-restricted MiHA HA-1H were isolated from HA-1Hneg/HLA-A*02:01pos and HA-1Hpos/HLA-A*02:01pos donors. Of the 16 unique HA-1H-specific T-cell clones, five T-cell clones derived from HA-1Hneg/HLA-A*02:01pos donors and one T-cell clone derived from an HA-1Hpos/HLA-A*02:01pos donor showed reactivity against HA-1Hpos target cells. In addition, in total, 663 T-cell clones (containing at least 91 unique clones expressing different T-cell receptors) directed against HLA*02:01-restricted peptides of TAA WT1-RMF, RHAMM-ILS, proteinase-3-VLQ, PRAME-VLD, and NY-eso-1-SLL were isolated from HLA-A*02:01pos donors. Only 3 PRAME-VLD-specific and one NY-eso-1-SLL-specific T-cell clone provoked interferon-γ production and/or cytolysis upon stimulation with HLA-A*02:01pos malignant cell lines (but not primary malignant samples) naturally overexpressing the TAA. These results show that self-HLA-restricted T cells specific for self-antigens such as MiHA in MiHApos donors and TAAs are present in peripheral blood of healthy individuals. However, clinical efficacy would require highly effective in vivo priming by peptide vaccination in the presence of proper adjuvants or in vitro expansion of the low numbers of self-antigen-specific T cells of sufficient avidity to recognize endogenously processed antigen.
Assuntos
Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Antígeno HLA-A2/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Linfócitos T Citotóxicos/imunologia , Humanos , Interferon gama/imunologia , Peptídeos/imunologiaRESUMO
BACKGROUND AIMS: To reduce the risk of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT), T-cell depletion (TCD) of grafts can be performed by the addition of alemtuzumab (ALT) "to the bag" (in vitro) before transplantation. In this prospective study, the authors analyzed the effect of in vitro incubation with 20 mg ALT on the composition of grafts prior to graft infusion. Furthermore, the authors assessed whether graft composition at the moment of infusion was predictive for T-cell reconstitution and development of GVHD early after TCD alloSCT. METHODS: Sixty granulocyte colony-stimulating factor-mobilized stem cell grafts were obtained from ≥9/10 HLA-matched related and unrelated donors. The composition of the grafts was analyzed by flow cytometry before and after in vitro incubation with ALT. T-cell reconstitution and incidence of severe GVHD were monitored until 12 weeks after transplantation. RESULTS: In vitro incubation of grafts with 20 mg ALT resulted in an initial median depletion efficiency of T-cell receptor (TCR) α/ß T cells of 96.7% (range, 63.5-99.8%), followed by subsequent depletion in vivo. Graft volumes and absolute leukocyte counts of grafts before the addition of ALT were not predictive for the efficiency of TCR α/ß T-cell depletion. CD4pos T cells were depleted more efficiently than CD8pos T cells, and naive and regulatory T cells were depleted more efficiently than memory and effector T cells. This differential depletion of T-cell subsets was in line with their reported differential CD52 expression. In vitro depletion efficiencies and absolute numbers of (naive) TCR α/ß T cells in the grafts after ALT incubation were not predictive for T-cell reconstitution or development of GVHD post- alloSCT. CONCLUSIONS: The addition of ALT to the bag is an easy, fast and generally applicable strategy to prevent GVHD in patients receiving alloSCT after myeloablative or non-myeloablative conditioning because of the efficient differential depletion of donor-derived lymphocytes and T cells.
Assuntos
Alemtuzumab/farmacologia , Transplante de Células-Tronco Hematopoéticas , Reconstituição Imune , Depleção Linfocítica/métodos , Subpopulações de Linfócitos T/efeitos dos fármacos , Adulto , Antineoplásicos Imunológicos/farmacologia , Doença Enxerto-Hospedeiro/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Subpopulações de Linfócitos T/fisiologiaRESUMO
Adult B-lymphoblastic leukemia (B-ALL) is a hematological malignancy characterized by genetic heterogeneity. Despite successful remission induction with classical chemotherapeutics and novel targeted agents, enduring remission is often hampered by disease relapse due to outgrowth of a pre-existing subclone resistant against the treatment. In this study, we show that small glycophosphatidylinositol (GPI)-anchor deficient CD52-negative B-cell populations are frequently present already at diagnosis in B-ALL patients, but not in patients suffering from other B-cell malignancies. We demonstrate that the GPI-anchor negative phenotype results from loss of mRNA expression of the PIGH gene, which is involved in the first step of GPI-anchor synthesis. Loss of PIGH mRNA expression within these B-ALL cells follows epigenetic silencing rather than gene mutation or deletion. The coinciding loss of CD52 membrane expression may contribute to the development of resistance to alemtuzumab (ALM) treatment in B-ALL patients resulting in the outgrowth of CD52-negative escape variants. Additional treatment with 5-aza-2'-deoxycytidine may restore expression of CD52 and revert ALM resistance.
Assuntos
Linfócitos B/metabolismo , Antígeno CD52/deficiência , Metilação de DNA/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Glicosilfosfatidilinositóis/deficiência , Proteínas de Membrana/genética , Proteínas de Neoplasias/deficiência , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Alemtuzumab/uso terapêutico , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Linfócitos B/patologia , Antígeno CD52/biossíntese , Antígeno CD52/genética , Linhagem Celular Tumoral , Decitabina/farmacologia , Decitabina/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Glicosilfosfatidilinositóis/biossíntese , Glicosilfosfatidilinositóis/genética , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genéticaRESUMO
After allogeneic stem cell transplantation (alloSCT), patient-derived stem cells that survived the pretransplantation conditioning compete with engrafting donor stem cells for bone marrow (BM) repopulation. In addition, donor-derived alloreactive T cells present in the stem cell product may favor establishment of complete donor-derived hematopoiesis by eliminating patient-derived lymphohematopoietic cells. T cell-depleted alloSCT with sequential transfer of potentially alloreactive T cells by donor lymphocyte infusion (DLI) provides a unique opportunity to selectively study how competitive repopulation and allo-immunologic pressure influence lymphohematopoietic recovery. This study aimed to determine the relative contribution of competitive repopulation and donor-derived anti-recipient alloimmunologic pressure on the establishment of lymphohematopoietic chimerism after alloSCT. In this retrospective cohort study of 281 acute leukemia patients treated according to a protocol combining alemtuzumab-based T cell-depleted alloSCT with prophylactic DLI, we investigated engraftment and quantitative donor chimerism in the BM and immune cell subsets. DLI-induced increase of chimerism and development of graft-versus-host disease (GVHD) were analyzed as complementary indicators for donor-derived anti-recipient alloimmunologic pressure. Profound suppression of patient immune cells by conditioning sufficed for sustained engraftment without necessity for myeloablative conditioning or development of clinically significant GVHD. Although 61% of the patients without any DLI or GVHD showed full donor chimerism (FDC) in the BM at 6 months after alloSCT, only 24% showed FDC in the CD4+ T cell compartment. In contrast, 75% of the patients who had received DLI and 83% of the patients with clinically significant GVHD had FDC in this compartment. In addition, 72% of the patients with mixed hematopoiesis receiving DLI converted to complete donor-derived hematopoiesis, of whom only 34% developed clinically significant GVHD. Our data show that competitive repopulation can be sufficient to reach complete donor-derived hematopoiesis, but that some alloimmunologic pressure is needed for the establishment of a completely donor-derived T cell compartment, either by the development of GVHD or by administration of DLI. We illustrate that it is possible to separate the graft-versus-leukemia effect from GVHD, as conversion to durable complete donor-derived hematopoiesis following DLI did not require induction of clinically significant GVHD.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Leucemia , Humanos , Linfócitos T , Quimerismo , Estudos Retrospectivos , Transplante Homólogo , Transfusão de Linfócitos/métodos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Doença Enxerto-Hospedeiro/prevenção & controleRESUMO
To broaden the applicability of adoptive T-cell therapy for the treatment of hematologic malignancies, we aim to start a clinical trial using HA-1-TCR transferred virus-specific T cells. TCRs directed against the minor histocompatibility antigen (MiHA) HA-1 are good candidates for TCR gene transfer to treat hematologic malignancies because of the hematopoiesis-restricted expression and favorable frequency of HA-1. For optimal anti-leukemic reactivity, high cell-surface expression of the introduced TCR is important. Previously, however, we have demonstrated that gene transferred HA-1-TCRs are poorly expressed at the cell-surface. In this study several strategies were explored to improve expression of transferred HA-1-TCRs.
Assuntos
Genes Codificadores dos Receptores de Linfócitos T/genética , Antígenos de Histocompatibilidade Menor/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Genes Codificadores dos Receptores de Linfócitos T/imunologia , Terapia Genética/métodos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/terapia , Humanos , Imunoterapia Adotiva/métodos , Antígenos de Histocompatibilidade Menor/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Retroviridae/genética , Retroviridae/imunologia , Linfócitos T/imunologia , Transdução GenéticaRESUMO
Prophylactic infusion of selected donor T cells can be an effective method to restore specific immunity after T-cell-depleted allogeneic stem cell transplantation (TCD-alloSCT). In this phase I/II study, we aimed to reduce the risk of viral complications and disease relapses by administrating donor-derived CD8pos T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus antigens, tumor-associated antigens (TAA) and minor histocompatibility antigens (MiHA). Twenty-seven of thirty-six screened HLA-A*02:01pos patients and their CMVpos and/or EBVpos donors were included. Using MHC-I-Streptamers, 27 T-cell products were generated containing a median of 5.2 × 106 cells. Twenty-four products were administered without infusion-related complications at a median of 58 days post alloSCT. No patients developed graft-versus-host disease during follow-up. Five patients showed disease progression without coinciding expansion of TAA/MiHA-specific T cells. Eight patients experienced CMV- and/or EBV-reactivations. Four of these reactivations were clinically relevant requiring antiviral treatment, of which two progressed to viral disease. All resolved ultimately. In 2/4 patients with EBV-reactivations and 6/8 patients with CMV-reactivations, viral loads were followed by the expansion of donor-derived virus target-antigen-specific T cells. In conclusion, generation of multi-antigen-specific T-cell products was feasible, infusions were well tolerated and expansion of target-antigen-specific T cells coinciding viral reactivations was illustrated in the majority of patients.
Assuntos
Neoplasias Hematológicas/terapia , Transplante de Células-Tronco , Linfócitos T/imunologia , Infecções por Adenoviridae/prevenção & controle , Adulto , Idoso , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/citologia , Infecções por Citomegalovirus/prevenção & controle , Infecções por Vírus Epstein-Barr/prevenção & controle , Estudos de Viabilidade , Feminino , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/imunologia , Humanos , Imunoterapia , Masculino , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/imunologia , Segurança do Paciente , Transplante HomólogoRESUMO
The aim of adoptive T-cell therapy of cancer is to selectively confer immunity against tumor cells. Autoimmune side effects, however, remain a risk, emphasizing the relevance of a suicide mechanism allowing in vivo elimination of infused T cells. We investigated the use of human CD20 as suicide gene in T-lymphocytes. Potential effects of forced CD20 expression on T-cell function were investigated by comparing CD20- and mock-transduced cytomegalovirus (CMV) specific T cells for cytolysis, cytokine release and proliferation. The use of CD20 as suicide gene was investigated in CMV specific T cells and in T cells genetically modified with an antigen specific T-cell receptor. No effect of CD20 on T-cell function was observed. CD20-transduced T cells with and without co-transferred T-cell receptor were efficiently eliminated by complement dependent cytotoxicity induced by therapeutic anti-CD20 antibody rituximab. The data support the broad value of CD20 as safety switch in adoptive T-cell therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD20/biossíntese , Antineoplásicos/farmacologia , Genes Transgênicos Suicidas , Imunoterapia Adotiva , Neoplasias/terapia , Retroviridae , Linfócitos T/transplante , Transdução Genética , Anticorpos Monoclonais Murinos , Antígenos CD20/imunologia , Linhagem Celular , Humanos , Neoplasias/imunologia , Neoplasias/metabolismo , Rituximab , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Administration of alemtuzumab (targeting the CD52 antigen) to the patient (in-vivo) or to the graft (in-vitro) before allogeneic stem cell transplantation (alloSCT) decreases the incidence of graft-versus-host disease (GvHD). Effectiveness of this treatment relies on depletion of donor T cells. Currently, no data are available on alemtuzumab pharmacokinetics and pharmacodynamics in patients who received combined in-vivo and in-vitro alemtuzumab-based T-cell depletion. In this prospective study, we analyzed alemtuzumab pharmacokinetics and its effect on the circulating T cells in 36 patients who received an allogeneic T-cell-depleted graft by addition of 20â¯mg alemtuzumab "to the bag" with or without prior alemtuzumab (30â¯mg cumulative dose intravenously) as part of the conditioning regimen. Effective T-cell depletion was shown for all patients, even though alemtuzumab plasma levels varied considerably. Peak alemtuzumab levels were observed directly after graft infusion and were not associated with the number of circulating T cells pre-infusion, but with plasma volumes of the patients. All patients engrafted, confirming feasibility of this transplantation protocol. Only three patients with low alemtuzumab levels developed acute GvHD (grade II in 2 patients and grade III in 1 patient). Persistence of circulating alemtuzumab at 3â¯weeks after transplantation had prevented reconstitution of CD52-positive T cells when alemtuzumab plasma levels were above 0.7⯵g/mL. However, overall T-cell reconstitution did not correlate with the levels of alemtuzumab exposure, due to early reconstitution of CD52-negative alemtuzumab-resistant T cells. The protective effect of these cells likely explains the low incidence of Epstein-Barr-virus- and cytomegalovirus-related disease despite circulating alemtuzumab.
Assuntos
Alemtuzumab/farmacocinética , Aloenxertos/efeitos dos fármacos , Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Infecções por Vírus Epstein-Barr/imunologia , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Herpesvirus Humano 4/fisiologia , Linfócitos T/imunologia , Adulto , Idoso , Alemtuzumab/uso terapêutico , Antígeno CD52/metabolismo , Células Cultivadas , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Estudos de Viabilidade , Feminino , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Ativação Linfocitária , Depleção Linfocítica , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Tolerância ao Transplante , Transplante Homólogo , Ativação Viral/efeitos dos fármacosRESUMO
Under non-inflammatory conditions HLA class II is predominantly expressed on hematopoietic cells. Therefore, donor CD4 T-cells after allogeneic stem cell transplantation (alloSCT) may mediate graft-vs.-leukemia reactivity without graft-vs.-host disease (GVHD). We analyzed immune responses in four patients converting from mixed to full donor chimerism without developing GVHD upon purified CD4 donor lymphocyte infusion (DLI) from their HLA-identical sibling donor after T-cell depleted alloSCT. In vivo activated T-cells were clonally isolated after CD4 DLI. Of the alloreactive T-cell clones, 96% were CD4 positive, illustrating the dominant role of CD4 T-cells in the immune responses. We identified 9 minor histocompatibility antigens (MiHA) as targets for alloreactivity, of which 8 were novel HLA class II restricted MiHA. In all patients, MiHA specific CD4 T-cells were found that were capable to lyse hematopoietic cells and to recognize normal and malignant cells. No GVHD was induced in these patients. Skin fibroblasts forced to express HLA class II, were recognized by only two MiHA specific CD4 T-cell clones. Of the 7 clones that failed to recognize fibroblasts, two targeted MiHA were encoded by genes not expressed in fibroblasts, presentation of one MiHA was dependent on HLA-DO, which is absent in fibroblasts, and T-cells recognizing the remaining 4 MiHA had an avidity that was apparently too low to recognize fibroblasts, despite clear recognition of hematopoietic cells. In conclusion, purified CD4 DLI from HLA-identical sibling donors can induce conversion from mixed to full donor chimerism with graft-vs.-malignancy reactivity, but without GVHD, by targeting HLA class II restricted MiHA.
Assuntos
Linfócitos T CD4-Positivos/transplante , Doença Enxerto-Hospedeiro/prevenção & controle , Leucemia/terapia , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Condicionamento Pré-Transplante/métodos , Linfócitos T CD4-Positivos/imunologia , Quimerismo , Feminino , Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Leucemia/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Doadores Vivos , Masculino , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/imunologia , Agonistas Mieloablativos/uso terapêutico , Irmãos , Transplante Homólogo , Resultado do TratamentoRESUMO
To broaden the applicability of cellular immunotherapy, adoptive transfer of T-cell receptor (TCR) transferred T cells may be an attractive strategy. Using this approach, high numbers of defined antigen-specific T cells can be engineered. As the introduced TCR has to compete for cell surface expression with the endogenous TCR, the introduced TCR chains are under control of a strong viral promotor, which, in contrast to the endogenous promotor, is constitutively active. We examined whether this difference in regulation would result in differences in TCR internalization and re-expression of the introduced and endogenous TCR on dual TCR engineered T cells and the antigen-responsiveness of both the TCRs. We showed comparable TCR downregulation of TCRs expressed under regulation of a retroviral promotor or the endogenous promotor. However, the introduced TCRs were rapidly re-expressed on the cell surface after TCR stimulation. Despite rapid re-expression of the introduced TCR, T cells exerted similar antigen-sensitivity compared with control T cells, showing that cell mechanisms other than TCR cell surface expression are involved in antigen-sensitivity directly after antigen-specific stimulation. These results showed that TCR transduced T cells are functionally not different from nontransduced T cells and can potentially be used as an effective treatment strategy.