RESUMO
BACKGROUND: Non-tumor-derived circulating DNA (nt-cirDNA) of advanced non-small cell lung cancer (NSCLC), with unclear origination, is associated with prognosis. We hypothesized that a part of nt-cirDNA release from CD3 or CD8 tumor-infiltrating lymphocytes (TILs) could have clinical implications. METHOD: To investigate the feasibility of T-cell-derived circulating DNA (T-cirDNA) detection, real-time PCR with Taqman assay-specific rearranged TCRß CDR3 region was conducted in plasma of 103 advanced NSCLC. CD3 and CD8-specific immunohistochemistry from biopsy specimen, was reviewed by one blinded pathologist to the T-cirDNA results. Prognostic impact including demographic characteristics was integrated into the model. RESULTS: Circulating DNA was detectable in 100 patients with median of 4 ng ml-1, while median of plasma T-cirDNA was 1.71 pg ml-1. Median %ratio of T-cirDNA/cirDNA was 0.02%. T-cirDNA was categorized by %ratio of T-cirDNA/cirDNA as undetectable, low (≤ 1%) and high (> 1%). Paradoxical prognostic impact of T-cirDNA/cirDNA was observed. Undetectable and high T-cirDNA/cirDNA translated to independent favorable prognostic outcome, HR of 0.54 [95% CI 0.30-0.96] and 0.41 [95% CI 0.21-0.80], respectively. 43 patients were assessed for CD3/CD8 TILs and PD-L1. High intratumoral CD3/CD8 TILs but not stromal CD3 TILs was correlated with high T-cirDNA/cirDNA representing active T-lymphocyte activity to eliminate cancer cells. While the prognosis of undetectable T-cirDNA/cirDNA, represents inactivated naïve T-cell, was determined by the presence of EGFR mutation and had long durable response of EGFR inhibitors. CONCLUSION: T-cirDNA could be a novel biomarker representing adaptive immune resistance in NSCLC patients. Further exploration as a predictive biomarker for EGFR inhibitors in setting of EGFR mutation might be warranted.
Assuntos
Adenocarcinoma/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Ácidos Nucleicos Livres/sangue , Neoplasias Pulmonares/sangue , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos T/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Complexo CD3 , Antígenos CD8 , Linfócitos T CD8-Positivos/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Ácidos Nucleicos Livres/metabolismo , Feminino , Estado Funcional , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de SobrevidaRESUMO
OBJECTIVES: T-cell receptor (TCR) gene rearrangement studies are widely used for assessing T-cell clonality. The frequency and significance of clonal peaks restricted to TCR ß (TCRB) tube C are uncertain. We retrospectively reviewed 80 TCR studies performed on bone marrow/peripheral blood. METHODS: TCRB and TCR γ (TCRG) analyses were performed using BIOMED-2 primers. A peak was considered clonal or atypical if it was reproducible and 5× or more or 3× to 5× polyclonal background, respectively. RESULTS: TCRB analysis demonstrated 12 (15%) of 80 cases with one to four isolated peaks in tube C (>3×) with polyclonal pattern in tubes A and B. TCRG analysis was monoclonal in two cases (both definite T-cell neoplasms), polyclonal in four, and oligoclonal in six. Of the 10 cases without clone in TCRG, six had autoimmune disorder and none had T-cell neoplasm. CONCLUSIONS: Peaks restricted to TCRB tube C in the TCR analysis may be misleading, as it is often not indicative of an overt T-cell neoplasm.
Assuntos
Rearranjo Gênico do Linfócito T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T/genética , Linfoma de Células T/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Clonais , Estudos de Coortes , Primers do DNA/genética , Feminino , Humanos , Linfoma de Células T/genética , Linfoma de Células T/patologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Linfócitos T/patologia , Adulto JovemRESUMO
INTRODUCTION: Chronic hepatitis B (CHB) is still a public health problem and its mechanism remains unclear. In this study, we detect the skewness of T cell receptor beta chain variable gene (TCR Vß) in peripheral blood lymphocytes (PBL) and the liver infiltrating lymphocytes (LIL) of patients with CHB; and hope to provide information for further research on the pathogenic mechanism of CHB. MATERIAL AND METHODS: Fifteen patients with CHB, ten healthy volunteers and three patients with liver cysts were recruited as the subjects. The usage of TCR Vß of PBL and LIL were measured and compared; the associations of the TCR Vß usage of PBL with some hematological indices, including human leukocyte antigen (HLA) alleles, percents of CD4+ and CD8+ T cells, sera levels of HBV-DNA and IFN-γ, were analyzed. RESULTS: In PBL, Vß12 and Vß13.1 were the highest predominant usage genes which usage frequencies were all 46.7%; Vß23 was the key limited usage gene (40.0%). In LIL, the mainly predominant and limited usage gene was Vß13.1 (73.3%) and Vß23 (46.7%), respectively. About half of the patients with CHB with HLA-DR9 or HLA-DR12 showed the predominant usage of Vß5.2 or Vß13.2. In patients with CHB, the percentage of CD4+ T cells was 33.41 ± 5.39 %, that of CD8+ T cells was 28.67 ± 6.77 %; the concentration of IFN-γ was 182.52 ± 44.16 pg/mL. Compared to the healthy controls, there were significant differences for these data (P < 0.05). Neither ALT nor HBV-DNA was relative to the usage of TCR Vß. CONCLUSIONS: PBL and LIL share the common sknewness of TCR Vß genes, which probably relates to some hematological indices. However, the roles of such similarities and associations in the development of CHB need further study.
Assuntos
Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Hepatite B Crônica/genética , Hepatite B Crônica/imunologia , Região Variável de Imunoglobulina/genética , Fígado/imunologia , Linfócitos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Adulto , Estudos de Casos e Controles , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Feminino , Subtipos Sorológicos de HLA-DR/genética , Subtipos Sorológicos de HLA-DR/imunologia , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/virologia , Interações Hospedeiro-Patógeno , Humanos , Região Variável de Imunoglobulina/imunologia , Fígado/virologia , Linfócitos/virologia , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Objective: We investigated the proportion of Vß T cell receptor (TCR) gene expression in peripheral CD3+ lymphocytes in familial and non-familial systemic lupus erythematosus (SLE) patients. Method: The Vß TCR repertoire was studied in 14 families in which several members had SLE. The Vß TCR usage in SLE patients (n = 27) was compared with that in healthy members of these multiplex families (n = 47), in 37 sporadic SLE patients who had no relatives with SLE, and in 15 healthy unrelated controls. Vß TCR repertoire expression was studied by multiparameter flow cytometry with the use of an array of 24 different Vß TCR gene family-specific monoclonal antibodies. Results: We found the same Vß TCR expression profile in the comparisons between sporadic SLE and familial SLE cases and healthy relatives, which included increased expression of Vß 5.2, Vß 11 and Vß 16, and lower expression of Vß 3, Vß 4, Vß 7.1 and Vß 17. Interestingly, solely Vß 17 was differentially expressed among sporadic and familial SLE. Also, increased expression of Vß 9 was the hallmark among familial SLE (casesand h ealthy relatives) in comparison to controls. Conclusion: These results highlight the notion that the final profile of the Vß TCR repertoire seen in familial and non-familial SLE seems to arise from the interaction of genetic, environmental, and immunoregulatory factors. Furthermore, it may contribute to the immunologic abnormalities affecting relatives of SLE patients.
Objetivo: Se investigó la proporción de la expresión génica del receptor variable beta de células T (Vß TCR) en linfocitos periféricos CD3+ en pacientes con lupus eritematoso generalizado (LEG) familiar y no familiar. Método: El repertorio de Vß TCR se estudió en 14 familias que presentaban más de un miembro con LEG. El uso de Vß TCR en pacientes con LEG (n = 27) se comparó con el de los miembros sanos de estas familias (n = 47), con 37 pacientes con LEG esporádico y con 15 controles sanos. La expresión del repertorio de Vß TCR se estudió por citometría de flujo multiparamétrica utilizando un arreglo de 24 diferentes anticuerpos monoclonales específicos de genes familiares para Vß TCR. Resultados: Se encontró el mismo perfil de expresión en las comparaciones entre los casos de LEG esporádico y familiar, así como en los consanguíneos sanos de las familias multicasos, que incluía una expresión incrementada de Vß 5.2, Vß 11 y Vß 16, y una menor expresión de Vß 3, Vß4, Vß 7.1 y Vß 7. De manera interesante, solo Vß 17 se expresó de modo diferente entre casos familiares y esporádicos de LEG. Igualmente, la expresión incrementada de Vß 9 fue el distintivo entre los casos de LEG familiar (casos y consanguíneos sanos) y los controles sanos. Conclusiones: Estos resultados refuerzan la noción de que el perfil final del repertorio Vß TCR observado en LEG familiar y no familiar parece surgir de la interacción de factores genéticos, ambientales e inmunorreguladores, además de que pueden explicar las alteraciones inmunitarias que se observan en los consanguíneos sanos de pacientes con LEG.
Assuntos
Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Lúpus Eritematoso Sistêmico/sangue , Linfócitos T , Estudos de Casos e Controles , Feminino , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Humanos , Lúpus Eritematoso Sistêmico/genética , MasculinoRESUMO
BACKGROUND: Immunoglobulin-cytokine fusion molecules have been shown to be the new generation of immunomodulating agents in transplantation tolerance induction. In the present study, we tested whether immunoregulatory cytokine fusion proteins of IL-10/Fc, TGF-ß/Fc, or IL-2/Fc would enhance allogeneic bone marrow cell (BMC) engraftment and promote tolerance induction. METHODS: B6 (H2) mice were conditioned with anti-CD154 (MR1) and rapamycin (Rapa) plus 100 cGy total body irradiation (MR1/Rapa/100 cGy) and transplanted with allogeneic B10.D2 (H2) BMC. Recipients were treated with lytic IL-2/Fc, nonlytic IL-2/Fc, TGF-ß/Fc, or IL-10/Fc fusion proteins to promote chimerism to induce tolerance. RESULTS: Donor chimerism was achieved in 20% of recipients conditioned with MR1/Rapa/100 cGy. The addition of TGF-ß/Fc (5- or 10-day treatment) or nonlytic IL-2/Fc (10-day treatment) fusion proteins to the conditioning resulted in engraftment in nearly 100% of recipients. In contrast, lytic IL-2/Fc or IL-10/Fc had no effect. The combination of nonlytic IL-2/Fc and TGF-ß/Fc had a synergistic effect to promote engraftment and resulted in significantly higher donor chimerism compared with recipients conditioned with TGF-ß/MR1/Rapa/100 cGy. Engraftment was durable in the majority of chimeras and increased over time. The chimeras accepted donor skin grafts and promptly rejected third-party skin grafts. Moreover, specific T cell receptor-Vß5.½ and TCR-Vß11 clonal deletion was detected in host T cells in chimeras, suggesting central tolerance to donor alloantigens. CONCLUSIONS: Allogeneic BMC engraftment is enhanced with TGF-ß/Fc fusion protein treatment. TGF-ß/Fc and nonlytic IL-2/Fc exert a synergistic effect in promotion of alloengraftment and donor-specific transplant tolerance, significantly decreasing the minimum total body irradiation dose required.
Assuntos
Transplante de Medula Óssea , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunossupressores/farmacologia , Interleucina-2/farmacologia , Transplante de Pele , Fator de Crescimento Transformador beta/farmacologia , Quimeras de Transplante , Condicionamento Pré-Transplante/métodos , Tolerância ao Transplante/efeitos dos fármacos , Animais , Transplante de Medula Óssea/efeitos adversos , Células Cultivadas , Técnicas de Cocultura , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Rejeição de Enxerto/imunologia , Isoantígenos/imunologia , Camundongos Endogâmicos C57BL , Modelos Animais , Proteínas Recombinantes de Fusão/farmacologia , Sirolimo/farmacologia , Transplante de Pele/efeitos adversos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , Transplante Homólogo , Irradiação Corporal TotalRESUMO
The development of molecules specific for M. tuberculosis-infected cells has important implications, as these tools may facilitate understanding of the mechanisms regulating host pathogen interactions in vivo. In addition, development of new tools capable to targeting M. tuberculosis-infected cells may have potential applications to diagnosis, treatment, and prevention of tuberculosis (TB). Due to the lack of CD1b polymorphism, M. tuberculosis lipid-CD1b complexes could be considered as universal tuberculosis infection markers. The aim of the present study was to display on the PIII surface protein of m13 phage, a human αß single-chain T-cell receptor molecule specific for CD1b:2-stearoyl-3-hydroxyphthioceranoyl-2´-sulfate-α-α´-D-trehalose (Ac2SGL) which is a complex presented by human cells infected with M. tuberculosis. The results showed the pIII fusion particle was successfully displayed on the phage surface. The study of the recognition of the recombinant phage in ELISA and immunohistochemistry showed the recognition of CD1b:Ac2SGL complexes and cells in human lung tissue from a tuberculosis patient respectively, suggesting the specific recognition of the lipid-CD1b complex.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos CD1/imunologia , Técnicas de Visualização da Superfície Celular , Glicolipídeos/imunologia , Mycobacterium tuberculosis/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Tuberculose/imunologia , Bacteriófago M13 , Linhagem Celular , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas ViraisRESUMO
A resposta imune envolve a participação de subpopulações de células naïve, efetoras e de memória, as quais são funcionalmente distintas, e a atividade destas células depende do reconhecimento antigênico, que ocorre no contexto MHC-peptídeo-TCR. A manutenção destas subpopulações e a homeostasia do sistema imune são controlados, entre outros fatores, pela apoptose. No entanto, este processo de morte também tem sido relacionado à diversas patologias, sendo responsável pela modulação de eventos imunológicos, interferindo no curso da infecção. Em pacientes com leishmaniose cutânea (LC), apesar da indução preferencial de linfócitos T CD4(positivo), os linfócitos T CD8(positivo) estão envolvidos nos processos de progressão e controle da infecção. Neste contexto, a citometria de fluxo foi utilizada definir simultaneamente as subpopulações de linfócitos T CD8(positivo) naïve, efetora, de memória central e de memória efetora; a diversidade do repertório VBeta destes linfócitos; e a ocorrência de apoptose, na resposta imune da LC. Neste estudo foram analisadas amostras de sangue periférico, obtidas ex vivo de pacientes durante e após o tratamento, e de indivíduos sadios; e após cultivo in vitro frente a antígenos de Leishmania braziliensis. Os resultados mostram que a apoptose exerce um papel modulados, diminuindo a freqüência de linfócitos T CD8(positivo), o que parece contribuir para a persistência da lesão ativa, no décimo dia de tratamento; enquanto a cura clínica parece ser conseqüência da diminuição de apoptose nestes linfócitos T CD8(positivo) contribuindo para o restabelecimento da freqüência destas células. A análise da distribuição das subpopulações de linfócitos T CD8(positivo) revelou que a proporção de células naïve e efetoras é invertida ao longo do tratamento e do processo de resolução da lesão. Paralelamente, apesar do maior percentual de linfócitos T CD8(positivo) efetores observados em pacientes durante o tratamento, a ocorrência de apoptose nestas células pode comprometer uma resposta imune eficaz, estando associada à presença de lesão ativa ulceradaO caráter modulador que este processo de morte exerce na resposta imune em ambos os grupos de pacientes sugere que a ocorrência de apoptose, mais intensa na fase ativa da doença, esteja relacionada à manutenção da infecção. A reunião das análises de repertório VBeta nas diferentes subpopulações de linfócitos T CD8(positivo) mostraram uma indução oligoclonal na qual as células que expressam VBeta 9, VBeta 13.2 ou VBeta 23 podem ser os clones selecionados a se diferenciar em células de memória central após a cura clínica; enquanto que os clones que expressam a cadeia VBeta 22, parecem ser preferencialmente selecionados a se diferenciar em células de memória efetora, durante a fase ativa da doença. Os únicos clones de linfócitos T CD8(positivo) efetores que apresentaram uma expansão significativa foram aqueles que expressam a cadeia VBeta 12, a qual parece estar associada à resposta imune efetora desencadeada na fase ativa da LC. Sendo assim, a heterogeneidade entre o repertório VBeta das diferentes subpopulações de linfócitos T CD8(positivo) ressalta a importância de avaliar separadamente o repertório das células naïve, efetoras, de memória central e de memória efetora.
Assuntos
Apoptose , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Leishmaniose Cutânea , Linfócitos TRESUMO
OBJECTIVE: To compare the frequencies of variants of TCRBV20S1 and TCRBV3S1 gene segments in patients with systemic sclerosis (SSc) and in controls. The null allele (allele 2) of TCRBV20S1 is associated with reduced levels of Vbeta20+ T-cells in the peripheral blood, while allele 1 of TCRBV3S1 is related to a low frequency of Vbeta3.1+ T-cells. METHODS: One hundred thirty patients with SSc and 118 healthy volunteer controls were genotyped for TCRBV20S1, and 117 patients and 85 controls were genotyped for TCRBV3S1 variants by PCR-RFLP. Patients underwent clinical evaluation, serology, pulmonary function tests, high resolution computed tomography, and Doppler echocardiography. RESULTS: The genotypic frequencies of TCRBV20S1 were 0.46 (allele 1/allele 1), 0.43 (allele 1/allele 2), and 0.11 (allele 2/allele 2) in SSc patients; in controls the frequencies were 0.70, 0.26, and 0.04, respectively (p < 0.001). The Mantel-Haenszel odds ratio (stratified by race and sex) of the allele 2 carrier state was 3.88 (95% CI 1.94 to 7.75). The allelic and genotypic frequencies of the TCRBV3S1 gene segment did not differ significantly in patients and controls. However, among patients, allele 1 (TCRBV3S1) carriers had a higher prevalence of interstitial lung disease (adjusted p = 0.032). CONCLUSION: The null allele of the TCRBV20S1 and the allele 1 of TCRBV3S1 gene segments may be considered risk factors for the development of SSc and interstitial lung disease, respectively, suggesting a protective role of Vbeta20+ and Vbeta3.1+ cells in the pathogenic immune responses in SSc.
Assuntos
Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Predisposição Genética para Doença/genética , Doenças Pulmonares Intersticiais/genética , Polimorfismo de Nucleotídeo Único/genética , Esclerodermia Difusa/genética , Esclerodermia Limitada/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Frequência do Gene , Humanos , Doenças Pulmonares Intersticiais/complicações , Masculino , Pessoa de Meia-Idade , Razão de Chances , Esclerodermia Difusa/complicações , Esclerodermia Limitada/complicaçõesRESUMO
Non-manipulated inbred mouse strains constitutes an interesting model-system for in vivo studies on thymus ontogeny due to the possibility to observe the molecular events of the thymocyte maturation. In previous studies, using RT-PCR method, we have found that several immune system genes such as interleukins and MHC are differentially expressed during ontogeny of the thymus whose genes act as modulators of T-cell differentiation. To determine which other genes are modulated on a large-scale basis, we measured the levels of mRNA expression in mouse fetal thymus (14-17 days of gestation) by hybridization with cDNA microarrays containing 1,576 cDNA sequences derived from the IMAGE MTB library. T-cell maturation was monitored by detection of the T-cell receptor beta TRBV8.1-BD2.1 rearranged DNA segment. Each developmental phase of thymus, displayed a characteristic expression profile, as evaluated by the Cluster and Tree-View softwares. Genes differentially and significantly expressed were selected on the basis of significance analysis of the microarray data (SAM program). With the reclustering of only significantly expressed genes, it was possible to characterize the phases of thymus ontogeny, based on the differential profile of expression. Our method provided the detection of genes implicated in the cell signaling, such as the hematopoietic cell signal transducer gene, genes implicated in T-cell calcium influx (tyrosine phosphatase) and calcium signaling proteins (vesicle transport binding protein 3, proline rich Gla, casein kinase alpha 1 and Down syndrome homolog protein 1) and a gene important for the protein transport, including T-cell receptors chains, towards the cell membrane (Golgi SNAP receptor complex member 2). The results demonstrate that the cDNA microarray used to explore the gene expression was useful for understanding the modulation of several cell-signaling genes, including the calcium cascade pathway, which is important for individual stages of T-cell maturation and control of anergy during thymus ontogeny.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Hibridização Genética , Linfócitos T/metabolismo , Timo/metabolismo , Animais , Perfilação da Expressão Gênica , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética , Linfócitos T/citologia , Timo/citologia , Timo/embriologiaRESUMO
The ageing of the immune system (immunosenescence) is believed to be involved in both morbidity and mortality in elderly humans due to a higher incidence of infections, autoimmune diseases, cancers and other pathological situations. As any specific immune response involves recognition of antigens by T cells, the ability to develop a given immune response is also dependent on the T-cell repertoire available at a given time point. Different T-cell receptor beta variable segment (BV) (TCRBV) gene segment alleles have been associated with diseases in various human populations. In the present work we analysed the allelic frequencies of four biallelic polymorphisms in TCRBV gene segments (TCRBV3S1, TCRBV13S5, TCRBV13S6 and TCRBV18) in healthy elderly human subjects (80 years old or more) from the south of Brazil, where life expectancies reach similar levels to those observed in developed countries. Except for allele 2 of the TCRBV13S6 polymorphism, which was more frequent in elderly than in young individuals (P = 0.0105), there were no differences in allele or genotype frequencies between young and elderly individuals. The data suggest that there is no direct correlation between the TCRBV3S1, TCRBV13S5 and TCRBV18 polymorphisms analysed and healthy senescence in this particular group of elderly individuals. The higher frequency of TCRBV13S6 allele 2 in healthy elderly individuals should be confirmed in other samples to establish the significance of this finding.