抗结核治疗中特异性T细胞免疫反应动态分析及其临床意义
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摘要
目前,抗结核的化疗方案包括2个月的强化期与4个月的巩固期,而治疗过程中特异性细胞免疫应答随细菌载量改变呈现的动态变化特征有可能成为治疗疗效的生物标志物,并为个性化治疗方案提供理论依据。为此,我们收集了9例活动性结核病患者治疗前,治疗2、4、6个月的外周血样本,采用酶联免疫斑点测定法(enzyme-linked immune spot assay,ELISPOT)测定了不同治疗阶段结核特异性抗原ESAT-6和CFP-10刺激下PBMC中分泌IFN-γ的细胞数量,同时分析了抗原特异性Th1型细胞因子分泌水平以及多功能T细胞比例的动态变化特征。结果显示:随着治疗的深入,ESAT-6及CFP-10刺激患者PBMC后,释放IFN-γ的细胞数量显著下降(P<0.05)。CD4~+IFN-γ~+T淋巴细胞的比例在治疗前与治疗6个月时相比有下降趋势,且治疗后结果具有归一性;虽然多功能T细胞有重要抗结核作用,但在我们统计的患者治疗过程中的变化不明显。以上结果表明抗结核治疗过程中外周淋巴细胞抗原特异性释放IFN-γ的细胞数量与CD4~+IFN-γ~+T淋巴细胞的比例随着患者的病情好转呈现下降趋势,提示抗原特异性细胞免疫应答的动态变化谱与病原菌负荷下降存在密切关系,可能成为抗结核治疗过程中的免疫学标志物。
The current anti-tuberculosis(TB)chemotherapy includes a two months intensive period and a four months consolidate period.In the process of anti-tuberculosis treatment,the dynamic change of mycobacterial specific cellular immune response is supposed to be affected by the decrease in bacteria loads,which might become biomarkers for treatment and prognosis.In this study,9active TB patients have been tracked to monitor the mycobacterial antigen specific T cell responses during the treatment.Peripheral blood samples were collected before the treatment and at 2,4and 6months of the treatment.Antigen specific IFN-γsecreting cells were determined by enzyme-linked immune assay spots(enzyme-linked immune spot assay,ELISPOT).Meanwhile,antigen specific cytokine-producing Th1 cells were detected by intracellular cytokine staining and mono-and multifunctional T cells proportion were analyzed.Our results showed that along with treatment,the numbers of IFN-γsecreting cells significantly decreased from(213.1±40.40)/2.5×10~5 to(113.5±23.82)/2.5×10~5(P=0.0144)upon ESAT-6stimulation and from(106.7±22.54)/2.5×10~5 to(34.94±17.6)/2.5×10~5(P=0.0234)upon CFP-10 stimulation.This was consistent with the results from flow cytometry that the percentage of IFN-γ~+CD4~+T cells was decreased after 6-month treatment.However,TNF-α~+CD4~+T and IL-2~+CD4~+T sustained at similar levels after treatment when compared to non-treatment points,which suggested different roles of cytokines during anti-TB immune responses.Although multifunctional T cells are reported to play an important role in anti-TB response,there exhibited no obvious change during the process of treatment in our study.Taken together,our study demonstrated that antigen specific IFN-γrelease displayed a decrease tendency,which was largely due to the reduction of bacilli load after the treatment.It might become the immunological biomarkers in the process of anti-tuberculosis treatment.
The current anti-tuberculosis(TB)chemotherapy includes a two months intensive period and a four months consolidate period.In the process of anti-tuberculosis treatment,the dynamic change of mycobacterial specific cellular immune response is supposed to be affected by the decrease in bacteria loads,which might become biomarkers for treatment and prognosis.In this study,9active TB patients have been tracked to monitor the mycobacterial antigen specific T cell responses during the treatment.Peripheral blood samples were collected before the treatment and at 2,4and 6months of the treatment.Antigen specific IFN-γsecreting cells were determined by enzyme-linked immune assay spots(enzyme-linked immune spot assay,ELISPOT).Meanwhile,antigen specific cytokine-producing Th1 cells were detected by intracellular cytokine staining and mono-and multifunctional T cells proportion were analyzed.Our results showed that along with treatment,the numbers of IFN-γsecreting cells significantly decreased from(213.1±40.40)/2.5×10~5 to(113.5±23.82)/2.5×10~5(P=0.0144)upon ESAT-6stimulation and from(106.7±22.54)/2.5×10~5 to(34.94±17.6)/2.5×10~5(P=0.0234)upon CFP-10 stimulation.This was consistent with the results from flow cytometry that the percentage of IFN-γ~+CD4~+T cells was decreased after 6-month treatment.However,TNF-α~+CD4~+T and IL-2~+CD4~+T sustained at similar levels after treatment when compared to non-treatment points,which suggested different roles of cytokines during anti-TB immune responses.Although multifunctional T cells are reported to play an important role in anti-TB response,there exhibited no obvious change during the process of treatment in our study.Taken together,our study demonstrated that antigen specific IFN-γrelease displayed a decrease tendency,which was largely due to the reduction of bacilli load after the treatment.It might become the immunological biomarkers in the process of anti-tuberculosis treatment.
引文
[1]WHO.Global tuberculosis report 2016[M].Geneva:WHO press,2016.
[2]尤旭华,李桂琴.肺结核治疗现状及展望[J].临床进展,2012,20(5):10.
[3]Flynn JL,Chan J.Immunology of tuberculosis[J].Ann Revi Immunol,2001,19:93-129.
[4]Flynn JL,Ernst JD.Immune responses in tuberculosis[J].Curr Opin Immunol,2000,12(4):432-436.
[5]Forbes EK,Sander C,Ronan EO,et al.Multifunctional,high-level cytokine-producing Th1cells in the lung,but not spleen,correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice[J].J Immunol,2008,181(7):4955-4964.
[6]Wilkinson KA,Wilkinson RJ.Polyfunctional T cells in human tuberculosis[J].Eur J Immunol,2010,40(8):2139-2142.
[7]Sahiratmadja E,Alisjahbana B,De Boer T,et al.Dynamic changes in pro-and anti-inflammatory cytokine profiles and gamma interferon receptor signaling integrity correlate with tuberculosis disease activity and response to curative treatment[J].Infect Immun,2007,75(2):820-829.
[8]Meier T,Eulenbruch HP,Wrighton-Smith P,et al.Sensitivity of a new commercial enzyme-linked immunospot assay(T SPOT-TB)for diagnosis of tuberculosis in clinical practice[J].Euro J Clin Microbiol Infect Dis,2005,24(8):529-536.
[9]Mazurek GH,Jereb J,Lobue P,et al.Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection,United States[J].MMWR Recommend Rep,2005,54(RR-15):49-55.
[10]Ernst JD.The immunological life cycle of tuberculosis[J].Nat Revi Immunol,2012,12(8):581-591.
[11]Hinks TS,Varsani N,Godsiff DT,et al.High background rates of positive tuberculosis-specific interferon-gamma release assays in a low prevalence region of UK:a surveillance study[J].BMC Infect Dis,2012,12:339.
[12]Park IN,Shim TS.Qualitative and quantitative results of interferon-gamma release assays for monitoring the response to anti-tuberculosis treatment[J].Korean J Intern Med,2016,32(2):302-308.
[13]Cozmei C,Constantinescu D,Carasevici E,et al.Th1and Th2cytokine response in patients with pulmonary tuberculosis and health care workers occupationally exposed to M.tuberculosis[J].Revista medico-chirurgicala a Societatii de Medici si Naturalisti din Iasi,2007,111(3):702-709.
[14]Saito S,Nakano M.Nitric oxide production by peritoneal macrophages of Mycobacterium bovis BCG-infected or non-infected mice:regulatory role of T lymphocytes and cytokines[J].J Leuk Biol,1996,59(6):908-915.
[15]Jo EK,Kim HJ,Lim JH,et al.Dysregulated production of interferon-gamma,interleukin-4and interleukin-6in early tuberculosis patients in response to antigen 85Bof Mycobacterium tuberculosis[J].Scand J Immunol,2000,51(2):209-217.
[16]Dorhoi A,Kaufmann SH.Tumor necrosis factor alpha in mycobacterial infection[J].Sem Immunol,2014,26(3):203-209.
[17]Johnson L,Gough J,Spencer Y,et al.Immunohistochemical markers augment evaluation of vaccine efficacy and disease severity in bacillus Calmette-Guerin(BCG)vaccinated cattle challenged with Mycobacterium bovis[J].Veter Immunol Immunopathol,2006,111(3-4):219-229.
[18]Darrah PA,Patel DT,De Luca PM,et al.Multifunctional TH1cells define a correlate of vaccine-mediated protection against Leishmania major[J].Nat Med,2007,13(7):843-850.
[19]Caccamo N,Guggino G,Joosten SA,et al.Multifunctional CD4+T cells correlate with active Mycobacterium tuberculosis infection[J].Euro J Immunol,2010,40(8):2211-2220.
[20]Harari A,Rozot V,Bellutti Enders F,et al.Dominant TNF-alpha+Mycobacterium tuberculosis-specific CD4+T cell responses discriminate between latent infection and active disease[J].Nat Med,2011,17(3):372-376.
[2]尤旭华,李桂琴.肺结核治疗现状及展望[J].临床进展,2012,20(5):10.
[3]Flynn JL,Chan J.Immunology of tuberculosis[J].Ann Revi Immunol,2001,19:93-129.
[4]Flynn JL,Ernst JD.Immune responses in tuberculosis[J].Curr Opin Immunol,2000,12(4):432-436.
[5]Forbes EK,Sander C,Ronan EO,et al.Multifunctional,high-level cytokine-producing Th1cells in the lung,but not spleen,correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice[J].J Immunol,2008,181(7):4955-4964.
[6]Wilkinson KA,Wilkinson RJ.Polyfunctional T cells in human tuberculosis[J].Eur J Immunol,2010,40(8):2139-2142.
[7]Sahiratmadja E,Alisjahbana B,De Boer T,et al.Dynamic changes in pro-and anti-inflammatory cytokine profiles and gamma interferon receptor signaling integrity correlate with tuberculosis disease activity and response to curative treatment[J].Infect Immun,2007,75(2):820-829.
[8]Meier T,Eulenbruch HP,Wrighton-Smith P,et al.Sensitivity of a new commercial enzyme-linked immunospot assay(T SPOT-TB)for diagnosis of tuberculosis in clinical practice[J].Euro J Clin Microbiol Infect Dis,2005,24(8):529-536.
[9]Mazurek GH,Jereb J,Lobue P,et al.Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection,United States[J].MMWR Recommend Rep,2005,54(RR-15):49-55.
[10]Ernst JD.The immunological life cycle of tuberculosis[J].Nat Revi Immunol,2012,12(8):581-591.
[11]Hinks TS,Varsani N,Godsiff DT,et al.High background rates of positive tuberculosis-specific interferon-gamma release assays in a low prevalence region of UK:a surveillance study[J].BMC Infect Dis,2012,12:339.
[12]Park IN,Shim TS.Qualitative and quantitative results of interferon-gamma release assays for monitoring the response to anti-tuberculosis treatment[J].Korean J Intern Med,2016,32(2):302-308.
[13]Cozmei C,Constantinescu D,Carasevici E,et al.Th1and Th2cytokine response in patients with pulmonary tuberculosis and health care workers occupationally exposed to M.tuberculosis[J].Revista medico-chirurgicala a Societatii de Medici si Naturalisti din Iasi,2007,111(3):702-709.
[14]Saito S,Nakano M.Nitric oxide production by peritoneal macrophages of Mycobacterium bovis BCG-infected or non-infected mice:regulatory role of T lymphocytes and cytokines[J].J Leuk Biol,1996,59(6):908-915.
[15]Jo EK,Kim HJ,Lim JH,et al.Dysregulated production of interferon-gamma,interleukin-4and interleukin-6in early tuberculosis patients in response to antigen 85Bof Mycobacterium tuberculosis[J].Scand J Immunol,2000,51(2):209-217.
[16]Dorhoi A,Kaufmann SH.Tumor necrosis factor alpha in mycobacterial infection[J].Sem Immunol,2014,26(3):203-209.
[17]Johnson L,Gough J,Spencer Y,et al.Immunohistochemical markers augment evaluation of vaccine efficacy and disease severity in bacillus Calmette-Guerin(BCG)vaccinated cattle challenged with Mycobacterium bovis[J].Veter Immunol Immunopathol,2006,111(3-4):219-229.
[18]Darrah PA,Patel DT,De Luca PM,et al.Multifunctional TH1cells define a correlate of vaccine-mediated protection against Leishmania major[J].Nat Med,2007,13(7):843-850.
[19]Caccamo N,Guggino G,Joosten SA,et al.Multifunctional CD4+T cells correlate with active Mycobacterium tuberculosis infection[J].Euro J Immunol,2010,40(8):2211-2220.
[20]Harari A,Rozot V,Bellutti Enders F,et al.Dominant TNF-alpha+Mycobacterium tuberculosis-specific CD4+T cell responses discriminate between latent infection and active disease[J].Nat Med,2011,17(3):372-376.