Terapia com ciclofosfamida no lúpus eritematoso sistêmico: heteronegeidade no impacto sobre o sistema imunitário adaptativo
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Data
2024-02-28
Autores
Agustinelli, Renan de Almeida [UNIFESP]
Orientadores
Andrade, Luis Eduardo Coelho [UNIFESP]
Tipo
Tese de doutorado
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Introdução: Pacientes com lúpus eritematoso sistêmico (LES) são frequentemente tratados com drogas imunossupressoras para diminuir o processo inflamatório lesivo. Alguns pacientes tornam-se vulneráveis a infecções enquanto outros parecem tolerar bem o uso da terapia imunossupressora. Isso sugere heterogeneidade individual do sistema imunitários de pacientes com LES aos imunossupressores. Objetivo: Avaliar se pacientes com nefrite lúpica apresentam heterogeneidade na susceptibilidade do sistema imune adaptativo ao tratamento com ciclofosfamida. Materiais e métodos: Foram incluídos sessenta pacientes com nefrite lúpica e tratamento com ciclofosfamida EV mensal. Colheu-se sangue antes (Tempo T1) e 20 dias após a 4ª pulsoterapia (Tempo T2) para imunofenotipagem de linfócitos T e B e seus subtipos, ensaio de proliferação de linfócitos T e dosagem sérica de IgA, IgM e IgG e suas subclasses. Trinta e nove pacientes completaram os 4 meses. Foram selecionados quarenta controles saudáveis pareados por sexo e idade para comparação em T1. Células mononucleares do sangue periférico foram marcadas com CFSE e cultivadas a 37ºC/5%CO2 sob estímulo com fitohemaglutinina ou anti-CD3/CD28. A proliferação foi avaliada após 72 horas por citometria de fluxo. As imunoglobulinas séricas foram quantificadas através de método imunoturbidimétrico. Trinta pacientes receberam vacina polissacarídica antipneumocócica e reforço contra o toxoide tetânico após a 4ª pulsoterapia e foi realizada nova coleta de sangue após 1 mês para a pesquisa de anticorpos contra 7 sorotipos do pneumococo e contra o toxoide tetânico por ELISA para comparação com os níveis anteriores à pulsoterapia. Resultados: Pacientes apresentaram menor frequência absoluta de linfócitos do que os controles, além de menor frequência absoluta e relativa de linfócitos T (LT) CD4+ de memória central e frequência absoluta de LT CD8+ de memória efetor e LT Th1 e Th2. Pacientes também apresentaram menor frequência absoluta de linfócitos B (LB) totais e LB imaturos, transicionais, LB com e sem switch de memória e plasmablastos, além de menor frequência relativa de LB naïve maduros. Encontramos maior capacidade proliferativa de LT estimulados com anti-CD3/CD28, além maiores níveis de IgG1 e IgG3 e menores níveis de IgG2 e IgG4 em pacientes. Na avaliação temporal, houve aumento da frequência relativa de LT CD4+, acompanhado de elevação de LT CD4+ de memória central e redução da frequência relativa de LT CD8+ de memória efetor. Também observamos redução da frequência relativa e absoluta de LB totais e absoluta de LB naïve maduros e de memória atípicos, redução da proliferação de LT estimulado com anti-CD3/CD28, dos níveis séricos de IgG, e de IgG1, IgG2 e IgG4. Após vacina contra o pneumococo houve elevação de anticorpos contra todos os sorotipos, apesar de onze pacientes não terem apresentado a resposta vacinal esperada. De forma semelhante, houve elevação dos anticorpos anti-toxóide tetânico, porém quatorze pacientes não obtiveram proteção plena. Análise hierárquica de clusters deflagrou três clusters em relação às variações temporais dos parâmetros imunológicos analisados, sendo que um desses clusters se associou com a ocorrência de infecções. A característica mais marcante desse cluster foi a redução dos níveis de imunoglobulinas após o tratamento. Conclusão: Houve flagrante heterogeneidade interindividual em relação à evolução temporal dos parâmetros imunitários estudados ao longo do tratamento com ciclofosfamida. Dentre os parâmetros analisados, a variação temporal dos níveis de imunoglobulinas parece despontar como potencial marcador para identificar risco de infecções durante o tratamento.
Introduction: Patients with systemic lupus erythematosus (SLE) are often treated with immunossuppressive drugs in order to achieve disease control. Some patients are more vulnerable to infections while others seem to tolerate the use of immunossuppressive drugs. This fact suggests individual heterogeneity of the immune system of SLE patients to immunosuppressants. Objective: Assess whether patients with lupus nephritis have a variation in the susceptibility of the adaptive immune system to treatment with the use of cyclophosphamide. Methods: Sixty patients with lupus nephritis treated with Monthly IV cyclophosphamide were enrolled. Blood was collected before (Time T1) and 20 days after the 4th pulse therapy (Time T2) for T and B lymphocytes and their subtypes immunophenotyping, T lymphocyte proliferation assay and serum dosage of IgA, IgM and IgG and its subclasses. Thirty-nine patients completed the 4 months. Forty healthy controls matched by sex and age were selected for comparison at T1. Peripheral blood mononuclear cells were labeled with CFSE and cultured at 37ºC/5%CO2 under stimulation with phytohemagglutinin or anti-CD3/CD28. Proliferation was assessed after 72 hours by flow cytometry. Serum immunoglobulins were quantified using the immunoturbidimetric method. Thirty patients received pneumococcal polysaccharide vaccine and tetanus toxoid booster after the 4th pulse therapy and a new blood drop was performed after 1 month for the investigation of antibodies against 7 pneumococcal serotypes and against tetanus toxoid by ELISA for comparison with the baseline levels. Results: Patients with lupus nephritis had fewer total lymphocytes than controls, as well as a lower absolute and relative frequency of central memory and effector CD4+ T lymphocytes and a lower absolute frequency of effector memory CD8+ cells and Th1 and Th2 T lymphocytes. Patients also presented a lower absolute frequency of total B lymphocytes and immature and transitional B lymphocytes, B lymphocytes with and without memory switch and plasmablasts, in addition to a lower relative frequency of mature naïve B cells. We observed greater proliferative capacity of T lymphocytes stimulated with antiCD3/CD28 in patients, in addition to higher levels of IgG1 and IgG3 and lower levels of IgG2 and IgG4 than controls. Regarding temporal evaluation patients presented an increase in the relative frequency of CD4+ cells, due to an increase in central memory CD4+ and a reduction in the relative frequency of effector memory CD8+ cells. We also observed a reduction in the relative and absolute frequency of total B cells and absolute frequency of naïve mature and atypical memory B cells, reduction in the proliferative capacity of T cells stimulated with antiCD3/CD28, in serum IgG levels, followed by a reduction in IgG1, IgG2 and IgG4. After the polysaccharide vaccine against pneumococcus, there was an increase in antibodies against all serotypes, although eleven patients did not show the expected response. Similarly, there was an increase in anti-tetanus toxoid antibodies, but fourteen patients did not obtain full protection. Cluster analysis triggered three distinct clusters in relation to variations in the immunological parameters analyzed, only one of which was associated with infections and the most striking characteristic of this cluster was the reduction in serum levels of immunoglobulins. Conclusion: We observed a striking inter-individual heterogeneity in relation to the temporal evolution of the parameters studied during treatment with cyclophosphamide. The temporal variation in immunoglobulin levels seems to emerge as a potential marker to identify the risk of infections during treatment.
Introduction: Patients with systemic lupus erythematosus (SLE) are often treated with immunossuppressive drugs in order to achieve disease control. Some patients are more vulnerable to infections while others seem to tolerate the use of immunossuppressive drugs. This fact suggests individual heterogeneity of the immune system of SLE patients to immunosuppressants. Objective: Assess whether patients with lupus nephritis have a variation in the susceptibility of the adaptive immune system to treatment with the use of cyclophosphamide. Methods: Sixty patients with lupus nephritis treated with Monthly IV cyclophosphamide were enrolled. Blood was collected before (Time T1) and 20 days after the 4th pulse therapy (Time T2) for T and B lymphocytes and their subtypes immunophenotyping, T lymphocyte proliferation assay and serum dosage of IgA, IgM and IgG and its subclasses. Thirty-nine patients completed the 4 months. Forty healthy controls matched by sex and age were selected for comparison at T1. Peripheral blood mononuclear cells were labeled with CFSE and cultured at 37ºC/5%CO2 under stimulation with phytohemagglutinin or anti-CD3/CD28. Proliferation was assessed after 72 hours by flow cytometry. Serum immunoglobulins were quantified using the immunoturbidimetric method. Thirty patients received pneumococcal polysaccharide vaccine and tetanus toxoid booster after the 4th pulse therapy and a new blood drop was performed after 1 month for the investigation of antibodies against 7 pneumococcal serotypes and against tetanus toxoid by ELISA for comparison with the baseline levels. Results: Patients with lupus nephritis had fewer total lymphocytes than controls, as well as a lower absolute and relative frequency of central memory and effector CD4+ T lymphocytes and a lower absolute frequency of effector memory CD8+ cells and Th1 and Th2 T lymphocytes. Patients also presented a lower absolute frequency of total B lymphocytes and immature and transitional B lymphocytes, B lymphocytes with and without memory switch and plasmablasts, in addition to a lower relative frequency of mature naïve B cells. We observed greater proliferative capacity of T lymphocytes stimulated with antiCD3/CD28 in patients, in addition to higher levels of IgG1 and IgG3 and lower levels of IgG2 and IgG4 than controls. Regarding temporal evaluation patients presented an increase in the relative frequency of CD4+ cells, due to an increase in central memory CD4+ and a reduction in the relative frequency of effector memory CD8+ cells. We also observed a reduction in the relative and absolute frequency of total B cells and absolute frequency of naïve mature and atypical memory B cells, reduction in the proliferative capacity of T cells stimulated with antiCD3/CD28, in serum IgG levels, followed by a reduction in IgG1, IgG2 and IgG4. After the polysaccharide vaccine against pneumococcus, there was an increase in antibodies against all serotypes, although eleven patients did not show the expected response. Similarly, there was an increase in anti-tetanus toxoid antibodies, but fourteen patients did not obtain full protection. Cluster analysis triggered three distinct clusters in relation to variations in the immunological parameters analyzed, only one of which was associated with infections and the most striking characteristic of this cluster was the reduction in serum levels of immunoglobulins. Conclusion: We observed a striking inter-individual heterogeneity in relation to the temporal evolution of the parameters studied during treatment with cyclophosphamide. The temporal variation in immunoglobulin levels seems to emerge as a potential marker to identify the risk of infections during treatment.
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Citação
AGUSTINELLI, Renan de Almeida. Terapia com ciclofosfamida no lúpus eritematoso sistêmico: heteronegeidade no impacto sobre o sistema imunitário adaptativo. 2024. 141 f. Tese (Doutorado em Ciências Reumatologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.