Ann Hematol (2014) 93:761–767 DOI 10.1007/s00277-013-1953-4
ORIGINAL ARTICLE
Immune status of Fanconi anemia patients: decrease in T CD8 and CD56dim CD16+ NK lymphocytes Graça A. Justo & Marco A. Bitencourt & Ricardo Pasquini & Morgana T. L. Castelo-Branco & Aline Almeida-Oliveira & Hilda Rachel Diamond & Vivian M. Rumjanek
Received: 14 January 2013 / Accepted: 4 November 2013 / Published online: 16 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Fanconi anemia (FA), a rare genetic disease in which patients' life is compromised mainly by hematological abnormalities and cancer prone, seems to be affected by subtle immune cell irregularities. Knowing that FA presents developmental abnormalities and, based on recent reports, suggesting that natural killer (NK) CD56dim and NK CD56bright correspond to sequential differentiation pathways, we investigated if there were changes on the total number of NK cells and subsets as well as on T CD4 and T CD8 lymphocytes and their ratio. A large sample of FA patients (n =42) was used in this work, and the results were correlated to clinical G. A. Justo Departamento de Bioquímica, IBRAG, UERJ, Rio de Janeiro, Brazil G. A. Justo : V. M. Rumjanek Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro, Brazil M. A. Bitencourt : R. Pasquini Ambulatório de Anemia de Fanconi, Hospital de Clínicas, Universidade Federal do Paraná, Curitiba, PR, Brazil M. T. L. Castelo-Branco Departamento de Histologia e Embriologia, ICB-CCS, UFRJ, Rio de Janeiro, Brazil A. Almeida-Oliveira : H. R. Diamond Centro de Transplante de Medula Óssea (CEMO), Instituto Nacional do Câncer (INCA), Rio de Janeiro, RJ, Brazil A. Almeida-Oliveira Fundação Oswaldo Cruz (Fiocruz), Instituto de Tecnologia Imunobiológica (Bio-Manguinhos), Rio de Janeiro, RJ, Brazil G. A. Justo (*) Laboratório de Imunologia Aplicada, Bioquímica de Proteínas e Produtos Naturais, Departamento de Bioquímica, Instituto de Biologia Alcântara Gomes, Universidade Estadual do Rio de Janeiro, Boulevard 28 de Setembro 87, Vila Isabel, Rio de Janeiro, Brazil 20551030 e-mail: [email protected]
hematological status of these patients. Among FA patients, a decreased proportion of T CD8+ and NK CD56dimCD16+ cells were observed when compared to healthy controls as well as an imbalance of the subsets NK lymphocytes. Data suggest that FA patients might have a defective cytotoxic response due to the lower number of cytotoxic cells as well as impairment in the differentiation process of the NK cells subsets which may be directly related to impairment of the immune surveillance observed in these patients. Keywords Fanconi anemia . Lymphocytes . Immune system . NK cells . CD8 T cells . Flow cytometry
Introduction Fanconi anemia (FA) is a rare autosomal recessive and Xlinked disorder. FA patients present congenital malformations, bone marrow failure, and increased propensity to cancer, especially acute myeloid leukemia [1–7]. In healthy subjects, FA proteins work on the FA/BRCA pathway to regulate homologous recombination repair during S phase or following DNA damage by cross-linking agents and double-strand breaks. Besides that, FA proteins protect cells from genotoxic stress, protecting hematopoietic cells from apoptosis, inhibiting proapoptotic signals, and activating the prosurvival ones [8]. The FA proteins seem also to be involved in many biochemical pathways associated with immune function, but little is known about this [9, 10]. To date, FA lymphocytes and/or their products inhibit the myeloid lineage cell growth, which may be related to the observed process of bone marrow failure [11]. Preliminary clinical studies showed FA patients' immune system irregularities, such as TNF-α upregulation, low levels of total lymphocytes, and increased susceptibility to bacterial infections [7, 10, 12, 13]. In a study with 10 FA patients, Fagerlie and Bagby [11] observed a diminished number of
762
both CD4+ and CD8+ T lymphocytes. A normal number of natural killer (NK) cells in FA patients have been described by Hersey et al. [10]; however, these cells exhibited low cytotoxic activity. Contrary to this result, Myers and co-workers [14], who studied 10 FA children, verified that the absolute number of natural killer cells as well as B lymphocytes was reduced, while those of T cells were within normal range. Recently, a similar result was obtained by Korthof et al. [15]. The NK and CD8 T cells are the main lymphocyte populations in the immune system with cytotoxic activity [16]. NK cells have an important function in the host response to infections and malignant cell clearance, linking innate and adaptive immunity [16]. In the peripheral blood, these cells totalize 10 % of lymphocytes divided into two subpopulations characterized by CD56 expression in the plasma membrane: CD56dim (~90 %) and CD56bright (~10 %). The CD56dim cells are highly cytotoxic and are found mainly in the peripheral blood. This subpopulation may express the FCγ III receptor (CD16), and it is involved on antibody-mediated cellular cytotoxicity (ADCC) [16]. On the other hand, CD56bright cells produce cytokines (IFN-γ, TNF-α, or IL-10) and chemokines after activation by macrophages, being mainly present in tissues and lymph nodes [17, 18], exhibiting immune regulatory functions. Triggers for cytokine production by CD56dim and CD56bright also differ. Cytokine production by NK CD56dim is triggered after target cell recognition, whereas the production by the NK CD56bright subpopulation is triggered via activation by other cytokines [17]. Furthermore, there are reports suggesting that CD56dim and CD56bright cell populations correspond to sequential differentiation pathways, in which NK CD56dim cell subset is the more differentiated one [19–21]. The balance between NK cell subsets is altered in several diseases; for instance, CD56dim subset is reduced in HIV patients [21, 22], and an increase of CD56bright was observed in lupus erythematosus [23]. Furthermore, treatment with IFN-α and IFN-β can lead to an increase in the CD56bright NK cell subset, as demonstrated by Saraste et al. [24] and Lee et al. [25]. Knowing that FA patients present developmental abnormalities and altered levels of TNF-α and INF-γ, we questioned if the number of NK lymphocytes was altered and/or if there were changes in the ratio of NK subsets, especially CD56dim and CD56bright sequentially differentiated populations. In addition, we measured the levels of T CD4 and T CD8 lymphocytes as well as CD4/CD8 ratio in these patients' blood samples. This study involved a large sample of FA patients, comprising 42 subjects, and data were compared to the clinical hematological status of the patient. Our results showed a decrease in the proportion of the two main immune cytotoxic cells T CD8 and NK CD56dimCD16+ when compared to healthy controls as well as an imbalance between the main subsets of NK lymphocytes CD56dim and CD56bright. Data suggest that FA patients may have their cytotoxic
Ann Hematol (2014) 93:761–767
lymphocytic function impaired due to the lower number of cytotoxic cells as well as a defect in the differentiation process of the NK cell subsets leading to a disability of the immune surveillance in these patients.
Methods FA patients and control subjects Peripheral blood mononuclear cells (PBMC) were obtained from 42 FA (ranging from 4 to 27 years old)-diagnosed patients [26] from the Clinical Hospital of Curitiba, Brazil, after the ethical approval by the National System of Research Ethics Committee–SISNEP (Brazil, FR16996). The FA patients were clinically evaluated in respect to their bone marrow cellularity as being normal (N) and with bone marrow hypocellularity (H) at the time of blood collection. PBMCs of 48 healthy volunteers/subjects (ranging from 2 to 40 years old) were included as controls. All samples (15 ml of peripheral blood) were collected with written informed consent. PBMC isolation Whole blood samples were previously submitted for red blood cell lyses by treatment with plasmim (1:8 v/v) (Sigma, St. Louis, MO, USA). PBMC cells were then separated using the Ficoll–Hypaque gradient (Histopaque1077, Sigma, St. Louis, MO, USA), washed once, and pelleted in RPMI (Sigma, St. Louis, MO, USA), containing 10 % heat-inactivated FCS (Gibco, Grand Island, NY, USA), 2 mM L -glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). T CD4+, T CD8+, NK, and NK subsets by flow cytometry analysis Samples containing 5 × 10 5–1 × 10 6 cells were washed in phosphate-buffered saline (PBS) plus 5 % human serum. The T CD4+ and T CD8+ lymphocyte subpopulations were quantified by direct immunofluorescence by incubation with mouse antihuman CD4+ fluorescein isothiocyanate (FITC) (Southern Biotechnology Associates, Inc., USA) and mouse antihuman CD8+ peridinin chlorophyll protein (PerCP) (BD Biosciences Pharmingen, USA) for 30 min at 4 °C. NK and NK subset cells were measured by indirect staining with mouse antihuman CD56 monoclonal antibody (Southern Biotechnology Associates, Inc., USA) and direct staining with mouse antihuman CD16 PE (BD Biosciences Pharmingen, USA). First, cells were incubated for 30 min at 4 °C with mouse antihuman CD56-purified monoclonal antibody, followed by washing once in PBS plus 5 % human serum, and incubated for 30 min at 4 °C with the secondary antibody FITC-labeled goat anti-mouse Ig (1:500 v/v) (BD Biosciences Pharmingen, USA). Cells were washed again and then incubated with anti-CD16 PE for 30 min at 4 °C. The final PBSwashed cell suspension was pelleted and resuspended in PBS for analysis by flow cytometry. Fluorescence data were acquired
Ann Hematol (2014) 93:761–767
on the FACSCalibur Flow Cytometer (Becton & Dickinson, San Jose, CA, USA). Data on a minimum of 104 cells were analyzed using the Summit v4.3 software (Dako Colorado, Inc., USA). For all measurements, a gate was defined for the lymphocyte population only, as exemplified in Fig. 1a. Statistical analysis Data were analyzed using the Mann– Whitney test and Dunnett T3 nonparametric tests with Spearman's rho comparisons test using SPSS 10.0 software for Windows (Chicago, IL, USA). A p value of
ORIGINAL ARTICLE
Immune status of Fanconi anemia patients: decrease in T CD8 and CD56dim CD16+ NK lymphocytes Graça A. Justo & Marco A. Bitencourt & Ricardo Pasquini & Morgana T. L. Castelo-Branco & Aline Almeida-Oliveira & Hilda Rachel Diamond & Vivian M. Rumjanek
Received: 14 January 2013 / Accepted: 4 November 2013 / Published online: 16 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Fanconi anemia (FA), a rare genetic disease in which patients' life is compromised mainly by hematological abnormalities and cancer prone, seems to be affected by subtle immune cell irregularities. Knowing that FA presents developmental abnormalities and, based on recent reports, suggesting that natural killer (NK) CD56dim and NK CD56bright correspond to sequential differentiation pathways, we investigated if there were changes on the total number of NK cells and subsets as well as on T CD4 and T CD8 lymphocytes and their ratio. A large sample of FA patients (n =42) was used in this work, and the results were correlated to clinical G. A. Justo Departamento de Bioquímica, IBRAG, UERJ, Rio de Janeiro, Brazil G. A. Justo : V. M. Rumjanek Instituto de Bioquímica Médica, CCS, UFRJ, Rio de Janeiro, Brazil M. A. Bitencourt : R. Pasquini Ambulatório de Anemia de Fanconi, Hospital de Clínicas, Universidade Federal do Paraná, Curitiba, PR, Brazil M. T. L. Castelo-Branco Departamento de Histologia e Embriologia, ICB-CCS, UFRJ, Rio de Janeiro, Brazil A. Almeida-Oliveira : H. R. Diamond Centro de Transplante de Medula Óssea (CEMO), Instituto Nacional do Câncer (INCA), Rio de Janeiro, RJ, Brazil A. Almeida-Oliveira Fundação Oswaldo Cruz (Fiocruz), Instituto de Tecnologia Imunobiológica (Bio-Manguinhos), Rio de Janeiro, RJ, Brazil G. A. Justo (*) Laboratório de Imunologia Aplicada, Bioquímica de Proteínas e Produtos Naturais, Departamento de Bioquímica, Instituto de Biologia Alcântara Gomes, Universidade Estadual do Rio de Janeiro, Boulevard 28 de Setembro 87, Vila Isabel, Rio de Janeiro, Brazil 20551030 e-mail: [email protected]
hematological status of these patients. Among FA patients, a decreased proportion of T CD8+ and NK CD56dimCD16+ cells were observed when compared to healthy controls as well as an imbalance of the subsets NK lymphocytes. Data suggest that FA patients might have a defective cytotoxic response due to the lower number of cytotoxic cells as well as impairment in the differentiation process of the NK cells subsets which may be directly related to impairment of the immune surveillance observed in these patients. Keywords Fanconi anemia . Lymphocytes . Immune system . NK cells . CD8 T cells . Flow cytometry
Introduction Fanconi anemia (FA) is a rare autosomal recessive and Xlinked disorder. FA patients present congenital malformations, bone marrow failure, and increased propensity to cancer, especially acute myeloid leukemia [1–7]. In healthy subjects, FA proteins work on the FA/BRCA pathway to regulate homologous recombination repair during S phase or following DNA damage by cross-linking agents and double-strand breaks. Besides that, FA proteins protect cells from genotoxic stress, protecting hematopoietic cells from apoptosis, inhibiting proapoptotic signals, and activating the prosurvival ones [8]. The FA proteins seem also to be involved in many biochemical pathways associated with immune function, but little is known about this [9, 10]. To date, FA lymphocytes and/or their products inhibit the myeloid lineage cell growth, which may be related to the observed process of bone marrow failure [11]. Preliminary clinical studies showed FA patients' immune system irregularities, such as TNF-α upregulation, low levels of total lymphocytes, and increased susceptibility to bacterial infections [7, 10, 12, 13]. In a study with 10 FA patients, Fagerlie and Bagby [11] observed a diminished number of
762
both CD4+ and CD8+ T lymphocytes. A normal number of natural killer (NK) cells in FA patients have been described by Hersey et al. [10]; however, these cells exhibited low cytotoxic activity. Contrary to this result, Myers and co-workers [14], who studied 10 FA children, verified that the absolute number of natural killer cells as well as B lymphocytes was reduced, while those of T cells were within normal range. Recently, a similar result was obtained by Korthof et al. [15]. The NK and CD8 T cells are the main lymphocyte populations in the immune system with cytotoxic activity [16]. NK cells have an important function in the host response to infections and malignant cell clearance, linking innate and adaptive immunity [16]. In the peripheral blood, these cells totalize 10 % of lymphocytes divided into two subpopulations characterized by CD56 expression in the plasma membrane: CD56dim (~90 %) and CD56bright (~10 %). The CD56dim cells are highly cytotoxic and are found mainly in the peripheral blood. This subpopulation may express the FCγ III receptor (CD16), and it is involved on antibody-mediated cellular cytotoxicity (ADCC) [16]. On the other hand, CD56bright cells produce cytokines (IFN-γ, TNF-α, or IL-10) and chemokines after activation by macrophages, being mainly present in tissues and lymph nodes [17, 18], exhibiting immune regulatory functions. Triggers for cytokine production by CD56dim and CD56bright also differ. Cytokine production by NK CD56dim is triggered after target cell recognition, whereas the production by the NK CD56bright subpopulation is triggered via activation by other cytokines [17]. Furthermore, there are reports suggesting that CD56dim and CD56bright cell populations correspond to sequential differentiation pathways, in which NK CD56dim cell subset is the more differentiated one [19–21]. The balance between NK cell subsets is altered in several diseases; for instance, CD56dim subset is reduced in HIV patients [21, 22], and an increase of CD56bright was observed in lupus erythematosus [23]. Furthermore, treatment with IFN-α and IFN-β can lead to an increase in the CD56bright NK cell subset, as demonstrated by Saraste et al. [24] and Lee et al. [25]. Knowing that FA patients present developmental abnormalities and altered levels of TNF-α and INF-γ, we questioned if the number of NK lymphocytes was altered and/or if there were changes in the ratio of NK subsets, especially CD56dim and CD56bright sequentially differentiated populations. In addition, we measured the levels of T CD4 and T CD8 lymphocytes as well as CD4/CD8 ratio in these patients' blood samples. This study involved a large sample of FA patients, comprising 42 subjects, and data were compared to the clinical hematological status of the patient. Our results showed a decrease in the proportion of the two main immune cytotoxic cells T CD8 and NK CD56dimCD16+ when compared to healthy controls as well as an imbalance between the main subsets of NK lymphocytes CD56dim and CD56bright. Data suggest that FA patients may have their cytotoxic
Ann Hematol (2014) 93:761–767
lymphocytic function impaired due to the lower number of cytotoxic cells as well as a defect in the differentiation process of the NK cell subsets leading to a disability of the immune surveillance in these patients.
Methods FA patients and control subjects Peripheral blood mononuclear cells (PBMC) were obtained from 42 FA (ranging from 4 to 27 years old)-diagnosed patients [26] from the Clinical Hospital of Curitiba, Brazil, after the ethical approval by the National System of Research Ethics Committee–SISNEP (Brazil, FR16996). The FA patients were clinically evaluated in respect to their bone marrow cellularity as being normal (N) and with bone marrow hypocellularity (H) at the time of blood collection. PBMCs of 48 healthy volunteers/subjects (ranging from 2 to 40 years old) were included as controls. All samples (15 ml of peripheral blood) were collected with written informed consent. PBMC isolation Whole blood samples were previously submitted for red blood cell lyses by treatment with plasmim (1:8 v/v) (Sigma, St. Louis, MO, USA). PBMC cells were then separated using the Ficoll–Hypaque gradient (Histopaque1077, Sigma, St. Louis, MO, USA), washed once, and pelleted in RPMI (Sigma, St. Louis, MO, USA), containing 10 % heat-inactivated FCS (Gibco, Grand Island, NY, USA), 2 mM L -glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). T CD4+, T CD8+, NK, and NK subsets by flow cytometry analysis Samples containing 5 × 10 5–1 × 10 6 cells were washed in phosphate-buffered saline (PBS) plus 5 % human serum. The T CD4+ and T CD8+ lymphocyte subpopulations were quantified by direct immunofluorescence by incubation with mouse antihuman CD4+ fluorescein isothiocyanate (FITC) (Southern Biotechnology Associates, Inc., USA) and mouse antihuman CD8+ peridinin chlorophyll protein (PerCP) (BD Biosciences Pharmingen, USA) for 30 min at 4 °C. NK and NK subset cells were measured by indirect staining with mouse antihuman CD56 monoclonal antibody (Southern Biotechnology Associates, Inc., USA) and direct staining with mouse antihuman CD16 PE (BD Biosciences Pharmingen, USA). First, cells were incubated for 30 min at 4 °C with mouse antihuman CD56-purified monoclonal antibody, followed by washing once in PBS plus 5 % human serum, and incubated for 30 min at 4 °C with the secondary antibody FITC-labeled goat anti-mouse Ig (1:500 v/v) (BD Biosciences Pharmingen, USA). Cells were washed again and then incubated with anti-CD16 PE for 30 min at 4 °C. The final PBSwashed cell suspension was pelleted and resuspended in PBS for analysis by flow cytometry. Fluorescence data were acquired
Ann Hematol (2014) 93:761–767
on the FACSCalibur Flow Cytometer (Becton & Dickinson, San Jose, CA, USA). Data on a minimum of 104 cells were analyzed using the Summit v4.3 software (Dako Colorado, Inc., USA). For all measurements, a gate was defined for the lymphocyte population only, as exemplified in Fig. 1a. Statistical analysis Data were analyzed using the Mann– Whitney test and Dunnett T3 nonparametric tests with Spearman's rho comparisons test using SPSS 10.0 software for Windows (Chicago, IL, USA). A p value of
