卡介苗多糖核酸对哮喘大鼠淋巴液和血液CD4~+CD25~+Foxp3~+调节性T细胞的影响

目的:探讨卡介苗多糖核酸(BCG-PSN)对哮喘大鼠淋巴液和血液调节性T细胞数量及功能的影响。方法:将SD大鼠随机分为对照组、哮喘组和BCG-PSN组,分别收集不同时间点大鼠淋巴液和血液,采用流式细胞仪(FCM)检测CD4+CD25+Foxp3+调节性T细胞(CD4+CD25+Foxp3+Treg)百分率,酶联免疫吸附试验(ELISA)检测淋巴液和血浆白介素10(IL-10)和转录生长因子β1(TGF-β1)浓度。结果:各组在各时间点其淋巴液中CD4+CD25+Foxp3+Treg百分率、IL-10水平均较血液明显升高。哮喘组大鼠淋巴液和血液中CD4+CD25+Foxp3+Treg百分率、IL-10、TGF-β1浓度均较对照组显著降低(P0.05)。BCG-PSN组淋巴液和血液中CD4+CD25+Foxp3+Treg百分率和IL-10水平较哮喘组明显升高(P0.05),与对照组比较无显著性差异;而TGF-β水平在48小时较对照组和哮喘组明显升高(P0.05)。结论:哮喘大鼠淋巴液和血液存在明显CD4+CD25+Foxp3+Treg数量及功能不足。BCG-PSN可能通过增加哮喘大鼠外周血和淋巴液中CD4+CD25+Foxp3+Treg的数量及其产生IL-10和TGF-β水平,增强免疫抑制效应,从而发挥抑制哮喘炎症的作用。     

CD4~＋ CD25~＋ Foxp3~＋调节性T细胞与自身免疫性疾病

自身免疫性疾病系由于机体免疫系统失衡,产生针对自身组织的免疫应答并导致自身组织、器官损害的一类疾病。调节性T淋巴细胞（regulatory T cell,Treg）具有免疫应答低下和免疫抑制特性,在维持机体免疫耐受和免疫应答稳态方面具有非常重要的作用,Treg的异常与多种自身免疫性疾病有关[1]。Foxp3特异性表达于CD4＋CD25＋Treg细胞,与其发育、成熟以及抑制功能关系密切。但是目前关于该转录因子的表达调控机制却不清楚。本文拟就CD4＋CD25＋Foxp3 Treg细胞的研究进展及与多种自身免疫性疾病的关系作一综述。     

Foxp3在CD25+CD4+调节性T细胞发育和功能中作用的研究进展

CD25+CD4+调节性T细胞(Treg)是显性耐受的重要调节细胞,在免疫病理、自身免疫耐受的维持、针对病原体和肿瘤的免疫反应调节过程中发挥着关键的作用.Foxp3作为X染色体编码的叉头蛋白转录因子家族的一员,是Treg发育、分化和功能发挥所必不可少的.研究Foxp3调控机制及其在Treg生物功能中的作用对于Treg和显性耐受的研究具有极其重要的意义.     

Foxp3在CD25+CD4+调节性T细胞发育和功能中作用的研究进展

CD25+CD4+调节性T细胞(Treg)是显性耐受的重要调节细胞,在免疫病理、自身免疫耐受的维持、针对病原体和肿瘤的免疫反应调节过程中发挥着关键的作用.Foxp3作为X染色体编码的叉头蛋白转录因子家族的一员,是Treg发育、分化和功能发挥所必不可少的.研究Foxp3调控机制及其在Treg生物功能中的作用对于Treg和显性耐受的研究具有极其重要的意义.     

CD73 expression on extracellular vesicles derived from CD4+CD25+Foxp3+ T cells contributes to their regulatory function

CD4⁺CD25⁺Foxp3⁺ Treg cells maintain immunological tolerance. In this study, the possibility that Treg cells control immune responses via the production of secreted membrane vesicles, such as exosomes, was investigated. Exosomes are released by many cell types, including T cells, and have regulatory functions. Indeed, TCR activation of both freshly isolated Treg cells and an antigen‐specific Treg‐cell line resulted in the production of exosomes as defined morphologically by EM and by the presence of tetraspanin molecules LAMP‐1/CD63 and CD81. Expression of the ecto‐5‐nucleotide enzyme CD73 by Treg cells has been shown to contribute to their suppressive function by converting extracellular adenosine‐5‐monophosphate to adenosine, which, following interaction with adenosine receptors expressed on target cells, leads to immune modulation. CD73 was evident on Treg cell derived exosomes, accordingly when these exosomes were incubated in the presence of adenosine‐5‐monophosphate production of adenosine was observed. Most importantly, CD73 present on Treg cell derived exosomes was essential for their suppressive function hitherto exosomes derived from a CD73‐negative CD4⁺ T‐cell line did not have such capabilities. Overall our findings demonstrate that CD73‐expressing exosomes produced by Treg cells following activation contribute to their suppressive activity through the production of adenosine.     

CD4+CD25+Foxp3+T细胞在子痫前期中作用机制的研究

目的 探讨子痫前期(PE)患者外周血中CD4+CD25+Foxp3+T细胞及胎盘组织Foxp3的表达水平.方法 73例PE患者分为MPE组(轻度PE,38例)和SPE组(重度PE,35例),以正常的孕妇作为对照组;采用流式细胞术(FCM)检测外周血中CD4+CD25+Foxp3+T细胞的表达水平,采用免疫组化法(IHC)检测胎盘组织Foxp3的表达水平;将Foxp3与CD4+CD25+Foxp3+T、胎盘重量和阿氏(Apgar)评分进行Spearman相关性分析.结果 SPE组、MPE组外周血中CD4+CD25+Foxp3+T细胞表达水平分别为4.23±0.74％、6.58±0.8％,均低于对照组的7.01±0.95 ％(P＜0.05),SPE组外周血中CD4+CD25+Foxp3+T细胞表达水平低于MPE组(P ＜0.05);SPE组、MPE组胎盘组织中Foxp3的阳性表达率分别为28.57％、47.37％,均低于对照组的82.76％(P ＜0.05),SPE组胎盘组织中Foxp3的阳性表达率显著低于MPE组(P＜0.05);胎盘组织中Foxp3的阳性表达率与CD4+CD25+Foxp3+T细胞表达水平、胎盘重量及Apgar评分均呈正相关(P＜0.01).结论 PE与外周血中CD4+CD25+Foxp3+T细胞表达水平下降密切相关.     

Immature dendritic cells convert anergic nonregulatory T cells into Foxp3−IL‐10+ regulatory T cells by engaging CD28 and CTLA‐4

Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide‐specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL‐10^?Foxp3^?CTLA‐4⁺CD25^lowEgr2⁺ T cells could be converted into suppressive IL‐10⁺Foxp3^?CTLA‐4⁺CD25^highEgr2⁺ cells resembling type‐1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF‐β during anergy induction favored Foxp3⁺ Treg‐cell induction, while TGF‐β had little effect when added to the second stimulation. Expression of both CD28 and CTLA‐4 molecules on anergic T cells was required to allow their conversion into Tr1‐like cells. Suppressor activity was enabled via CD28‐mediated CD25 upregulation, acting as an IL‐2 sink, together with a CTLA‐4‐mediated inhibition of NFATc1/α activation to shut down IL‐2‐mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1‐like cells.     

CD4+CD25+ Treg induction by an HSP60‐derived peptide SJMHE1 from Schistosoma japonicum is TLR2 dependent

Chronic schistosome infection results in the suppression of host immune responses, allowing long‐term schistosome survival and restricting pathology. Current theories suggest that Treg play an important role in this regulation. However, the mechanism of Treg induction during schistosome infection is still unknown. The aim of this study was to determine the mechanism behind the induction of CD4⁺CD25⁺ T cells by Schistosoma japonicum HSP60 (SjHSP60)‐derived peptide SJMHE1 as well as to elucidate the cellular and molecular basis for the induction of CD4⁺CD25⁺ T cells during S. japonicum infection. Mice immunized with SJMHE1 or spleen and LN cells from naïve mice pretreated with SJMHE1 in vitro all displayed an increase in CD4⁺CD25⁺ T‐cell populations. Release of IL‐10 and TGF‐β by SJMHE1 stimulation may contribute to suppression. Adoptively transferred SJMHE1‐induced CD4⁺CD25⁺ T cells inhibited delayed‐type hypersensitivity in BALB/c mice. Additionally, SJMHE1‐treated APC were tolerogenic and induced CD4⁺ cells to differentiate into suppressive CD4⁺CD25⁺ Treg. Furthermore, our data support a role for TLR2 in SJMHE1‐mediated CD4⁺CD25⁺ Treg induction. These findings provide the basis for a more complete understanding of the S. japonicum–host interactions that contribute to host homeostatic mechanisms, preventing an excessive immune response.     

CD8+ Treg cells suppress CD8+ T cell‐responses by IL‐10‐dependent mechanism during H5N1 influenza virus infection

Although Treg‐cell‐mediated suppression during infection or autoimmunity has been described, functions of Treg cells during highly pathogenic avian influenza virus infection remain poorly characterized. Here we found that in Foxp3‐GFP transgenic mice, CD8⁺ Foxp3⁺ Treg cells, but not CD4⁺ Foxp3⁺ Treg cells, were remarkably induced during H5N1 infection. In addition to expressing CD25, the CD8⁺ Foxp3⁺ Treg cells showed a high level of GITR and produced IL‐10. In an adoptive transfer model, CD8⁺ Treg cells suppressed CD8⁺ T‐cell responses and promoted H5N1 virus infection, resulting in enhanced mortality and increased virus load in the lung. Furthermore, in vitro neutralization of IL‐10 and studies with IL‐10R‐deficient mice in vitro and in vivo demonstrated an important role for IL‐10 production in the capacity of CD8⁺ Treg cells to inhibit CD8⁺ T‐cell responses. Our findings identify a previously unrecognized role of CD8⁺ Treg cells in the negative regulation of CD8⁺ T‐cell responses and suggest that modulation of CD8⁺ Treg cells may be a therapeutic strategy to control H5N1 viral infection.     

不同结核病患者CD4+CD25+Foxp3+调节性T细胞表达的变化

Objective To investigate the expression of CD4 + CD25 + Foxp3 + regulatory T cell in patients with tuberculosis, and to discover its role in the immune response to mycobacterium tuberculosis. Methods Thirty-three patients with tuberculosis and 30 healthy controls were selected who were consulted in our hospital. Patients were classified by their chemotherapy and smear sputum and CD4 + CD25 + Foxp3 + regulatory T cell were detected by flow cytometry. Results Expression of CD4 + CD25high and CD4+ CD25 + Foxp3 + regulatory T cell in experimental group were ( 8.84 ± 2.55 ) % and (6.30 ± 1.38 ) % respectively, which were significantly higher than they were in control group (t = 3.57,4. 01, P ＜ 0. 01 ). The expression of CD4 + CD25high and CD4+ CD25 + Foxp3 + regulatory T cell in patients with positive smear sputum were also significant higher than that in patients with negative smear sputum ( t = 2. 51,2. 42,P ＜ 0.05). No significance was founded between the first-visit group and revisit group ( t = 0. 03, 0. 02, P ＞ 0.05 ). Conclusions CD4 + CD25high and CD4 + CD25 + Foxp3 + regulatory T cell in patients with tuberculosis were higher than healthy control, which may result in immune suppression.     

不明原因复发性流产小鼠模型CD4~+CD25~+ Treg的比例及Foxp3表达的变化

目的分析比较小鼠正常妊娠模型和流产模型中CD4+CD25+调节性T细胞CD4+CD25+Treg的比例、Foxp3蛋白表达水平及胎盘吸收率,探讨CD4+CD25+Treg与不明原因复发性流产的关系。方法以雌性CBA/J×雄性Balb/c为对照组,以雌性CBA/J×雄性DBA/2J为流产组,各10对,于妊娠第14天采用流式细胞术(flow cytometry,FCM)分析CD4+CD25+Treg在CD4+细胞中所占比例,Western blot检测并比较2组CD4+T细胞中Foxp3的表达,并观察2组小鼠的胎盘吸收情况。结果流产组CD4+CD25+Treg所占比例为(10.1±0.59)%低于对照组(15.5±0.78)%(P<0.05)。流产组胚胎吸收率为(25.6±3.5)%,明显高于对照组(2.4±1.6)%(P<0.01)。流产组Foxp3谱带密度相对值为0.30±0.018,低于正常组的0.68±0.025(P<0.05)。结论小鼠的自然流产模型建立成功,流产组孕鼠CD4+CD25+Treg的比例和Foxp3蛋白表达水平明显低于正常妊娠组,提示流产组高流产率可能与CD4+CD25+Treg数量和Foxp3的表达减少有关,CD4+CD25+Treg细胞的数量减少和功能缺陷可能是不明原因复发性流产的发生机制之一。     

CD8+ Foxp3+ T cells share developmental and phenotypic features with classical CD4+ Foxp3+ regulatory T cells but lack potent suppressive activity

"Suppressor T cells" were historically defined within the CD8(+) T-cell compartment and recent studies have highlighted several naturally occurring CD8(+) Foxp3(-) Treg populations. However, the relevance of CD8(+) Foxp3(+) T cells, which represent a minor population in both thymi and secondary lymphoid organs of nonmanipulated mice, remains unclear. We here demonstrate that de novo Foxp3 induction in peripheral CD8(+) Foxp3(-) T cells is counter-regulated by DC-mediated co-stimulation via CD80/CD86. CD8(+) Foxp3(+) T cells fail to develop in TCR-transgenic mice with Rag1(-/-) background, similar to classical CD4(+) Foxp3(+) Tregs. Notably, both naturally occurring and induced CD8(+) Foxp3(+) T cells express bona fide Treg markers including CD25, GITR, CTLA4 and CD103, and show defective IFN-γ production upon restimulation when compared with their CD8(+) Foxp3(-) counterparts. However, utilizing DEREG transgenic mice for the isolation of Foxp3(+) cells by eGFP reporter expression, we demonstrate that induced CD8(+) Foxp3(+) T cells similar to activated CD8(+) Foxp3(-) T cells only mildly suppress T-cell proliferation and IFN-γ production. We therefore categorize CD8(+) Foxp3(+) T cells as a tightly controlled population sharing certain developmental and phenotypic properties with classical CD4(+) Foxp3(+) Tregs, but lacking potent suppressive activity.     

CD4+CD25+Treg在急性淋巴细胞白血病患者外周血中的表达及其体外实验研究

目的初步探讨CD4 CD25 调节性T细胞(CD4 CD25 regulatory T cells,CD4 CD25 Treg)在急性淋巴细胞白血病(acute lymphocytic leukemia,ALL)患者化疗前及化疗缓解后外周血中的表达水平,并研究患者血清能否诱导外周血CD4 CD25-T细胞转化为CD4 CD25 Treg。方法①采用流式细胞术分别检测ALL初诊组、化疗完全缓解或部分缓解组及正常对照组外周血中CD4 CD25 T细胞所占比例,然后通过荧光定量RT-PCR检测各组外周血中转录因子Foxp3mRNA的表达水平,并逐层分析比较。②采集正常人外周血单个核细胞后,对照组用正常人血清,实验组用患者血清并分别设浓度梯度进行培养,72h后采用流式细胞术、荧光定量RT-PCR分别检测CD4 CD25 T细胞和Foxp3mRNA表达。结果ALL化疗缓解组CD4 CD25 T细胞及Foxp3mRNA表达水平均明显高于ALL初诊组和正常对照组(P<0.05),后两者之间CD4 CD25 T细胞水平无统计学差异(P>0.05),但ALL初诊组Foxp3mRNA含量较正常对照组明显升高(P<0.01),差异具有统计学意义;并且血清培养对照组CD4 CD25 T细胞水平及Foxp3mRNA含量均明显低于实验组(P<0.05),且其表达并不随血清浓度的增加而升高。结论CD4 CD25 Foxp3 Treg在ALL初诊组及化疗缓解组患者外周血中比例明显升高,且初步表明患者血清中的可溶性物质可诱导外周血CD4 CD25 T细胞转化为CD4 CD25 Treg,提示CD4 CD25 Treg可能是ALL免疫抑制的一个重要原因。     

IL‐12 and IL‐4 activate a CD39‐dependent intrinsic peripheral tolerance mechanism in CD8+ T cells

Immune responses to protein antigens involve CD4⁺ and CD8⁺ T cells, which follow distinct programs of differentiation. Naïve CD8 T cells rapidly develop cytotoxic T‐cell (CTL) activity after T‐cell receptor stimulation, and we have previously shown that this is accompanied by suppressive activity in the presence of specific cytokines, i.e. IL‐12 and IL‐4. Cytokine‐induced CD8⁺ regulatory T (Treg) cells are one of several Treg‐cell phenotypes and are Foxp3^? IL‐10⁺ with contact‐dependent suppressive capacity. Here, we show they also express high level CD39, an ecto‐nucleotidase that degrades extracellular ATP, and this contributes to their suppressive activity. CD39 expression was found to be upregulated on CD8⁺ T cells during peripheral tolerance induction in vivo, accompanied by release of IL‐12 and IL‐10. CD39 was also upregulated during respiratory tolerance induction to inhaled allergen and on tumor‐infiltrating CD8⁺ T cells. Production of IL‐10 and expression of CD39 by CD8⁺ T cells was independently regulated, being respectively blocked by extracellular ATP and enhanced by an A2A adenosine receptor agonist. Our results suggest that any CTL can develop suppressive activity when exposed to specific cytokines in the absence of alarmins. Thus negative feedback controls CTL expansion under regulation from both nucleotide and cytokine environment within tissues.     

Lewis肺癌细胞通过TLR9对CD4~+CD25~+Treg细胞影响的研究

目的:本研究以Lewis肺癌细胞为研究对象,探讨肿瘤细胞通过TLRs对CD4+CD25+Treg细胞的影响。方法:我们采用流式细胞术检测了Lewis肺癌细胞与脾淋巴细胞共培养系统中CD4+CD25+Treg细胞数量变化;通过RT-PCR方法检测了共培养对Foxp3和TLR1-9mRNA表达的影响;采用TLR9受体阻断剂氯喹阻断Lewis肺癌TLR9的表达。结果:与对照组相比,共培养组CD4+CD25+Treg细胞数量及Foxp3 mRNA表达均明显增高(P0.05);Lewis肺癌细胞与淋巴细胞共培养后可影响多种TLRs表达,其中TLR9 mRNA表达与对照组相比明显增高(P0.05),阻断Lewis肺癌细胞TLR9可明显降低CD4+CD25+Treg细胞数量及Foxp3 mRNA表达(P0.05)。结论:Lewis肺癌细胞可通过TLR9促进CD4+CD25+Treg细胞产生及功能增强,参与诱导肿瘤的免疫耐受,从而促进肿瘤的发生和发展。     

初发系统性红斑狼疮患者外周血CD4~+CD25~+Foxp3~+和CD4~+CD25~-Foxp3~+T细胞

目的：研究初发系统性红斑狼疮患者（Systemic lupus elythematosus，SLE）外周血CD4^＋T细胞中CD25和Foxp3表达及其在SLE发病中的意义。方法：根据SLE疾病活动积分（SLEDAI）将初发SLE患者分为活动组（10例）和不活动组（11例），流式细胞仪检测治疗前后外周血CD4^＋T细胞中CD25、Foxp3和CD127表达百分率，并对其与SLE临床活动度、尿蛋白、补体和anti-ds-DNA相关性进行研究。结果：初发活动组和不活动组SLE患者CD4^＋CD25^＋Foxp3^＋T细胞表达百分率分别为（1．91％～6．75％）和（2．74％～7．01％），与正常对照（2．11％～9．90％）相比没有统计学差异（P=0．524，P=0．794）；且初发SLE患者外周血CD4^＋CD25^＋T细胞在体外增殖反应和增殖抑制功能与正常对照相比无明显差别（P=0．174，P=0．689）；外周血CD4^＋CD25^-Foxp3^＋T细胞百分率在初发活动组（3．71％～10．94％）和不活动组（2．97％～7．69％）SLE患者均比正常对照（1．01％～3．62％）显著增高（P〈0．01和P〈0．01）；而CD4^＋CD2^＋Foxp34^-T百分率在初发活动组SLE患者（1．19％～9．23％）显著低于正常对照（2．67％～11．26％）和初发不活动组SLE患者（3．73～8．27％）（P=0．039，P=0．048）；与CD4^＋CD25^＋Foxp3^＋T细胞类似，90％左右的CD4^＋CD25一Foxp3^＋T细胞不表达或低表达CD127，其百分率与anti-ds-DNA浓度呈正相关，且尽管未达到统计学意义，但激素和免疫抑制治疗后其水平下降。结论：初发未经治疗的SLE患者CD4^＋CD25^＋Foxp3^＋T细胞数量和功能无明显异常，而CD4^＋CD25^-Foxp3^＋T细胞数量增多，与SLE疾病活动相关，可能具有调节功能。     

介导CD4+CD25+调节性T细胞发育与功能的一个关键基因--Foxp3

CD4^＋CD25^＋调节性T细胞是天然产生的调节性T细胞的重要亚群之一，在维持免疫系统稳态中发挥重要的作用。目前有关CD4^＋CD25^＋调节性T细胞分化发育的确切机制还不清楚。最近的研究发现Foxp3基因与CD4^＋CD25^＋调节性T细胞的分化发育和免疫调节功能的关系密切，进而提示Foxp3可能是CD4^＋CD25^＋调节性T细胞的一个特征性标志。     

介导CD4+CD25+调节性T细胞发育与功能的一个关键基因--Foxp3

CD4 CD25 调节性T细胞是天然产生的调节性T细胞的重要亚群之一,在维持免疫系统稳态中发挥重要的作用。目前有关CD4 CD25 调节性T细胞分化发育的确切机制还不清楚。最近的研究发现Foxp3基因与CD4 CD25 调节性T细胞的分化发育和免疫调节功能的关系密切,进而提示Foxp3可能是CD4 CD25 调节性T细胞的一个特征性标志。     

小鼠CD4~+ CD25~+ Foxp3~+调节性T细胞体外扩增

目的:本研究旨在探讨CD4+CD25+Foxp3+调节性T细胞体外扩增的方法。方法:采用磁珠分选小鼠CD4+T细胞,αCD3单克隆抗体包被24孔板,加入αCD28单克隆抗体、雷帕霉素、rhIL-2,培养3周后,流式细胞仪测定培养细胞中CD4+CD25+T细胞的含量,实时定量PCR检测CD4+CD25+T细胞Foxp3 mRNA的表达;单向混合淋巴细胞反应和增殖抑制试验测定扩增的CD4+CD25+T细胞的增殖及其抑制功能;ELISA检测培养上清中IL-10和TGF-β1的含量。结果:小鼠CD4+T细胞培养3周后,CD4+CD25+T细胞达(76.05±2.73)%,高于未加雷帕霉素组(52.17±1.36)%(P<0.001),磁珠分选的CD4+CD25+T细胞Foxp3 mRNA的表达是未加雷帕霉素组的5倍(P<0.001),增殖能力是未加雷帕霉素组的0.29倍(P<0.001),对CD4+T细胞增殖抑制能力是未加雷帕霉素组的3.6倍(P<0.001),培养上清中IL-10和TGF-β1分别是对照组的1.8倍和1.6倍(P<0.001)。结论:小鼠CD4+T细胞在含有1μg/ml的αCD28、rhIL-2 100 U/ml和终浓度为10 nmol/L雷帕霉素的培养体系中培养3周后能有效扩增CD4+CD25+Foxp3+调节性T细胞。     

Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4+/CD4+ Foxp3+ cells

CD4⁺ Foxp3⁺ regulatory T cells inhibit the production of interferon‐γ, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat‐shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4⁺ Foxp3⁺ cells compared with non‐immunized mice. Heterologous immunization using bacillus Calmette–Guérin (BCG) to prime and DNA‐hsp 65 to boost (BCG/DNA‐hsp 65) or BCG to prime and culture filtrate proteins (CFP)‐CpG to boost (BCG/CFP‐CpG) induced a significantly higher ratio of spleen CD4⁺/CD4⁺ Foxp3⁺ cells compared with non‐immunized mice. In addition, the protection conferred by either the BCG/DNA‐hsp 65 or the BCG/CFP‐CpG vaccines was significant compared with the DNA‐hsp 65 vaccine. Despite the higher ratio of spleen CD4⁺/CD4⁺ Foxp3⁺ cells found in BCG/DNA‐hsp 65‐immunized or BCG/CFP‐CpG‐immunized mice, the lungs of both groups of mice were better preserved than those of DNA‐hsp 65‐immunized mice. These results confirm the protective efficacy of BCG/DNA‐hsp 65 and BCG/CFP‐CpG heterologous prime‐boost vaccines and the DNA‐hsp 65 homologous vaccine. Additionally, the prime‐boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4⁺ and regulatory (CD4⁺ Foxp3⁺) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.