RESUMO
Dendritic cells (DC) are professional (specialised) antigen-presenting cells that can capture antigen from apoptotic tumour cells and induce MHC class I- and II-restricted responses. Also, DC fused with tumour cells may be effective for immune response induction. Both cell preparations may be considered as vaccine candidates in a therapeutic approach. We examined autologous T-cell activation by DC that had endocytosed leukaemic B-cell apoptotic bodies (Apo-DC) and compared it to the T-cell stimulatory capacity of DC that were fused with tumour cells. Following incubation, 22.6+/-6.2 (mean+/-s.e.m.) of DC had endocytosed leukaemic cells, while the frequency of DC-leukaemic cell hybrids was 10.5+/-2.6%. Apo-DC and hybrid cells both demonstrated the ability to stimulate a tumour-specific T-cell immune response in vitro. A T-cell proliferation response was also observed in four out of five CLL patients when using Apo-DC. However, fusion hybrids lacked the ability to elicit a proliferative response. Apo-DC also induced an IFN-gamma response, as did hybrid cells. The cytokine response induced by Apo-DC was significantly higher than that induced by fusion (P<0.05). This study shows that endocytosed apoptotic tumour cells induced a significantly stronger T-cell response than DC hybrids; and as such should be a better candidate for vaccine production.
Assuntos
Apoptose , Células Dendríticas/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apresentação de Antígeno , Antígenos CD/metabolismo , Linfócitos B/imunologia , Estudos de Casos e Controles , Divisão Celular , Fusão Celular , Quimera , Endocitose , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunofenotipagem , Imunoterapia , Interferon gama/metabolismo , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Pessoa de Meia-IdadeRESUMO
Little information is available on long-term immune reconstitution after therapy with alemtuzumab in B-CLL patients. We present long-term follow-up data for blood lymphocyte subsets analysed by flow cytometry in previously untreated B-CLL patients who received alemtuzumab subcutaneously as first-line therapy. All lymphoid subsets were significantly (P<0.001) and profoundly reduced; the median end-of-treatment counts for CD4(+), CD8(+), CD3(-)56(+) (natural killer (NK)), CD3(+)56(+) (NK-T) and CD19(+)5(-) (normal B) cells were 43, 20, 4, 1 and 8 cells/microl, respectively. The median cell count of all subsets remained at <25% of the baseline values for >9 months post-treatment. CD4(+) and CD8(+) levels in blood had reached >100 cells/microl in >50% of the patients at 4 months after the end of treatment. One patient had a cytomegalovirus reactivation and one patient developed Pneumocystis carinii pneumonia during therapy. No opportunistic or other major infections were recorded during unmaintained, long-term follow-up. There was no correlation between the cumulative dose of alemtuzumab and the severity or length of immunosuppression. CD52(-) T-cell subsets occurred during the treatment and comprised >80% of all CD4(+) and CD8(+) cells in the blood at the end of therapy. These subpopulations declined gradually during unmaintained follow-up. The relationship between these observations and the safety/antitumour effects of alemtuzumab is discussed.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antineoplásicos/administração & dosagem , Antineoplásicos/administração & dosagem , Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Alemtuzumab , Anticorpos Monoclonais Humanizados , Antígenos CD/imunologia , Estudos de Casos e Controles , Seguimentos , Humanos , Imunidade Celular , Imunofenotipagem , Injeções Subcutâneas , Pessoa de Meia-Idade , Indução de Remissão , Resultado do TratamentoRESUMO
TCRBV (T-cell receptor B variable) gene usage and CDR3 size distribution were analyzed using reverse transcription polymerase chain reaction (RT-PCR) to assess the T-cell repertoire of 10 patients with B-cell chronic lymphocytic leukemia (B-CLL) and in nine age-matched healthy control donors. When the usage of each TCRBV gene within the CD8(+) T cells of the patients was compared with that of the controls, no statistically significant difference was noted except for BV 6S1-3. In contrast, within the CD4(+) T cells of the CLL patients, a statistically significant overexpression for four BV families (2, 3, 5S1, 6S1-3) was seen while an underrepresentation was noted for five BV families (10, 11, 15, 16, 19). Based on the criterion that a value of any BV higher than the mean + 3 standard deviation (SD) of healthy controls indicated an overexpression, individual patients were shown to overexpress several TCRBV genes compared with the controls. Analyses of the CDR3 length polymorphism showed a significantly higher degree of restriction within CD4(+) and CD8(+) T cells of the patients, as compared with the corresponding control T-cell population. There was a significant difference in the CDR3 size distribution pattern with a more polymorphic CDR3 length pattern in the age-matched controls as compared with CLL patients, suggesting different mechanisms driving the T cells towards a clonal/oligoclonal TCRBV usage in patients and controls, respectively. The results show major perturbations of T cells in CLL patients, more frequently seen in the CD4(+) T-cell subset, indicating that nonmalignant CD4(+) T cells may be involved in the pathogenesis of CLL, but also CD8(+) T cells.
Assuntos
Linfócitos T CD4-Positivos/patologia , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Região Variável de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Dendritic cells (DC) are attractive candidates for use in vaccine-based immunotherapy. We have analysed the functional capability of DC generated in vitro from blood CD14(+) cells of chronic lymphocytic leukaemia (CLL) patients and healthy donors by culturing for 10 d with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4) and tumour necrosis factor-alpha (TNF-alpha). Two distinct DC populations were identified in patients as well as in controls. The majority of DC expressed CD11c and a minority also CD123. Most of the DC generated from both patients and controls exhibited a mature phenotype indicated by CD83 and major histocompatibility complex (MHC) class II expression, as well as by a characteristic morphology. Less than 1% of DC exhibited CD14. CLL DC had a similar expression of accessory molecules (CD54, CD80 and CD86) as control DC. The mean fluorescence intensity of CD80 and MHC class I molecules was significantly higher on CLL DC than on control DC (P < 0.05). At the gene level (real-time polymerase chain reaction) the expression of IL-10 was higher in CLL (P = 0.028) than in control DC. IL-1 beta and IL-12p(35) transcripts were also more abundant in CLL than in control DC but did not reach statistical significance. The expression of IL-4 and TNF-alpha was similar to that of control DC. The interferon gamma (IFN-gamma) gene expression level in CLL DC was decreased compared with control DC. DC of CLL patients had a similar capacity to stimulate in mixed leucocyte reaction as well as to present a recall antigen (PPD) as control DC. Thus, DC of CLL patients seem to have a normal function and may serve as antigen preserving cells for presentation of tumour antigens in a therapeutic vaccination approach. The mechanisms behind the observed increase in some surface molecules and the abnormal cytokine profile of CLL DC is not clear but might indicate pre-activation of DC in vivo, which may have a regulatory role in the pathobiology of CLL.
Assuntos
Células Dendríticas/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Idoso , Apresentação de Antígeno , Técnicas de Cultura de Células , Diferenciação Celular/imunologia , Citocinas/biossíntese , Células Dendríticas/patologia , Feminino , Humanos , Imunofenotipagem , Receptores de Lipopolissacarídeos/sangue , Teste de Cultura Mista de Linfócitos , Masculino , Reação em Cadeia da Polimerase/métodos , Linfócitos T/imunologiaRESUMO
We have previously shown that autologous T cells recognize leukaemic cells from patients with chronic lymphocytic leukaemia (B-CLL) in an MHC class I- and/or II-restricted manner. A candidate recognition structure might be the tumour cell-derived Ig VH complementarity-determining region (CDR)3. Three patients with B-CLL were analysed for the presence of autologous T cells recognizing the tumour-specific VH-CDR3 region. The VH region was shown to be mutated in all three patients. In two patients, a VH-CDR3-specific T-cell response was detected by proliferation assay, as well as by gamma-interferon (IFN) production. The responses could be inhibited by monoclonal antibodies against MHC class II, but not MHC class I. In the third patient, a VH-CDR3 proliferative response was detected, which could be inhibited by an anti-MHC class I monoclonal antibody, but not by anti-MHC class II antibodies. No gamma-IFN response could be detected in this patient. In no patient was an interleukin (IL)-4 response noted. Thus, in patients with B-CLL, naturally occurring T cells recognizing the tumour-unique VH-CDR3 region are present.
Assuntos
Genes de Imunoglobulinas , Leucemia Linfocítica Crônica de Células B/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Linfócitos T/imunologia , Idoso , Sequência de Aminoácidos , Anticorpos Monoclonais/farmacologia , Sequência de Bases , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunofenotipagem , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , MutaçãoRESUMO
This study analysed a naturally occurring specific cellular immunity against tumour cells in chronic lymphocytic leukaemia (CLL) patients. Five out of eight patients had blood T lymphocytes able to recognize spontaneously and specifically the autologous tumour B cells (proliferation assay). In these five patients, detection of cytokines by real-time reverse transcription polymerase chain reaction (RT-PCR) revealed that granulocyte-macrophage colony-stimulating factor (GM-CSF) was the most abundant cytokine gene expressed by the T cells that recognized the autologous tumour B cells. Other activated cytokine genes were gamma-interferon (IFN), interleukin (IL)-2 and tumour necrosis factor (TNF)-alpha, but not IL-4. This profile suggests a type 1 anti-B-CLL T-cell response. CD80 and CD54 were relatively downregulated on the native tumour B cells compared with control normal B cells. Upregulation of CD80 on the leukaemic cells was mandatory for the induction of such a specific T-cell response. CD80 and CD54 monoclonal antibodies inhibited the specific T-cell DNA synthesis proliferation. The proliferative T-cell response was either MHC class I or class II restricted (inhibition by monoclonal antibodies). The specific cytokine gene expression could be found in isolated CD4, as well as CD8, T-cell subsets. This study demonstrated the presence of a potential natural specific CD4, as well as a CD8 type 1 T-cell immunity against the leukaemic CLL tumour B cells in CLL. A further detailed analysis of the spontaneous anti-CLL T-cell immunity is warranted that may facilitate the development of effective anti-tumour vaccines in CLL.