Proliferation and apoptosis of lamina propria CD4+ T cells from scid mice with inflammatory bowel disease

Scid mice transplanted with low numbers of syngeneic CD4⁺ T cells, develop a chronic and lethal inflammatory bowel disease (IBD) within 4 – 6 months. We have used in vivo 5-bromo-2-deoxy-uridine (BrdU) labeling to assess the proliferation of lamina propria-derived CD4⁺ T cells in diseased scid mice. The hourly rate of renewal of colonic lamina propria CD4⁺ T cells in diseased mice was 7 % compared with 1.5 % in normal BALB/c control mice. Transplantation of scid mice with in vitro activated CD4⁺ T cells accelerated the disease onset and development in a cell dose-dependent fashion when compared with non-activated CD4⁺ T cells. In pulse-chase experiments it was shown that BrdU-labeled cells disappeared rapidly from the lamina propria of diseased mice. DNA analysis revealed that this was due to the presence of nearly four times as many apoptotic CD4⁺ T cells in diseased than in control mice. Further analyses showed that the apoptotic lamina propria CD4⁺ T cells were derived from cells having entered the cell cycle within the previous 8 h. These data clearly demonstrate that vigorous CD4⁺ T cell proliferation and death are involved throughout the course of IBD.     

CD4+CD25+调节性T细胞及相关细胞因子的研究进展

胸腺来源的CD4^＋CD25^＋调节性T细胞（Treg）是机体维持自身免疫耐受的重要组成部分，约占CD4^＋T细胞的5％-10％。它具有免疫抑制及免疫无能的特性，是最重要的Treg细胞的亚群之一。近年发现CD4^＋CD25^＋Treg细胞主要通过分泌一些抑制性细胞因子和抑制自身反应性T细胞的免疫应答等方式在维持自身免疫耐受中扮演着重要的角色，其数量的缺乏或功能紊乱会导致各种自身免疫性疾病的发生。     

CD4+ T regulatory cells from the colonic lamina propria of normal mice inhibit proliferation of enterobacteria-reactive,disease-inducing Th1-cells from scid mice with colitis

Adoptive transfer of CD4+ T cells into scid mice leads to a chronic colitis in the recipients. The transferred CD4+ T cells accumulate in the intestinal lamina propria (LP), express an activated Th1 phenotype and proliferate vigorously when exposed ex vivo to enteric bacterial antigens. As LP CD4+ T cells from normal BALB/c mice do not respond to enteric bacterial antigens, we have investigated whether colonic LP-derived CD4+ T cells from normal mice suppress the antibacterial response of CD4+ T cells from scid mice with colitis. LP-derived CD4+ T cells cocultured with bone marrow-derived dendritic cells effectively suppress the antibacterial proliferative response of CD4+ T cells from scid mice with colitis. The majority of these LP T-reg cells display a nonactivated phenotype and suppression is independent of antigen exposure, is partly mediated by soluble factor(s) different from IL-10 and TGF-beta, and is not prevented by the addition of high doses of IL-2 to the assay culture. Functionally and phenotypically the T-reg cells of the present study differ from previously described subsets of T-reg cells. The presence of T cells with a regulatory potential in the normal colonic mucosa suggests a role for these cells in the maintenance of local immune homeostasis of the gut.     

Lack of TNFR2 expression by CD4+ T cells exacerbates experimental colitis

TNF plays fundamental roles in the induction and perpetuation of inflammation. The effects of TNF are mediated through TNF receptor (TNFR) 1 or 2. As these two receptors mediate different functions, selective targeting of one receptor may represent a more specific treatment for inflammatory disorders than the complete blocking of TNF. TNFR2 expression is up‐regulated in inflammatory bowel disease. Hence, we directly assessed the role of TNFR2 signaling in the CD4⁺ T‐cell transfer model of colitis using TNFR2^?/? or WT mice as donors of colitogenic CD4⁺CD45RB^hi T cells for transfer into syngeneic RAG2^?/? or RAG2^?/?TNFR2^?/? recipient mice. Although the absence of TNFR2 expression by non‐lymphoid cells of the recipient mice does not influence the course of colitis, transfer of TNFR2^?/? CD4⁺ T cells leads to an accelerated onset of disease and to more severe signs of inflammation. The enhanced colitogenic potential of TNFR2^?/? CD4⁺ T cells is associated with reduced activation‐induced cell death, resulting in an increased accumulation of TNFR2^?/? CD4⁺ T cells. Hence, TNFR2 signaling is crucial for the TNF‐dependent contraction of the disease‐inducing T cells. Therefore, a selective blocking of TNFR2 may lead to exacerbation rather than attenuation of T‐cell‐mediated inflammatory disorders.     

Polyclonal expansion of adoptively transferred CD4+ αβ T cells in the colonic lamina propria of scid mice with colitis

The adoptive transfer of low numbers of peripheral, non-fractionated CD4⁺ αβ T cells into histocompatible, severely immunodeficient (scid) hosts induces a colitis. This disease developed in C.B-17 scid/scid hosts after the injection of 10⁵CD4⁺ T cells purified from different peripheral lymphoid organs of immunocompetent C.B-17 +/+ or BALB/c^dm2 donor mice. Irrespective of their tissue origin, transferred CD4⁺ T cells selectively repopulated the scid host with gut-seeking CD4⁺ T cells. A chronic inflammatory bowel disease (IBD) developed as polyclonal populations of mucosa-seeking memory/effector CD4⁺ T cells accumulated in the gut lamina propria and epithelial layer of the adoptive host. The manifestation of colitis in the scid host correlated with the in situ polyclonal activation and expansion of adoptively transferred CD4⁺ T cells in the colonic lamina propria. Attempts were unsuccessful to select in vivo an oligoclonal CD4⁺ T cell population with an enhanced IBD-inducing potential by repeatedly reinjecting 10⁵ donor-type CD4⁺ T cells from the colonic lamina propria of transplanted scid mice with an early and severe IBD into new scid hosts. The data indicate that the preferential repopulation of gut-associated lymphoid tissues with immunocompetent CD4⁺ T cells, and their polyclonal activation and in situ expansion in the lamina propria of the histocompatible, immunodeficient host are critical events in the pathogenesis of an IBD in this model.     

CD4+CD25+调节性T细胞在肿瘤免疫中的作用

CD4+CD25+调节性T细胞(Tr)是体内自然发生的调节性T细胞的重要亚群,具有无反应性和免疫抑制两大特性,主要通过与靶细胞的直接接触而起作用,其在体内不仅参与自身免疫性疾病、移植排斥反应等,还在肿瘤的发生、发展及免疫治疗中发挥重要作用.近几年来,Tr在肿瘤免疫中的作用倍受关注.     

FTY720 suppresses the development of colitis in lymphoid‐null mice by modulating the trafficking of colitogenic CD4+ T cells in bone marrow

2‐Amino‐2‐(2‐[4‐octylphenyl]ethyl)‐1,3‐propanediol hydrochloride (FTY720) suppresses T‐cell egress from LN, thereby preventing pathogenic T cells from migrating toward disease sites. However, little is known about whether FTY720 could control the trafficking of T cells without the presence of lymphoid tissues. Here we demonstrate that FTY720 treatment suppresses the recirculation of CD4⁺ T cells in splenectomized (SPX) lymphotoxin‐α^?/? (LT‐α^?/?) mice that lack LN and spleen, as shown by peripheral blood (PB) lymphopenia in FTY720‐treated SPX LT‐α^?/? mice. In a short‐term transfer experiment, the cell number of transferred Ly5.1⁺CD4⁺ T cells recovered from host FTY720‐treated SPX LT‐α^?/? mice (Ly5.2⁺) was markedly decreased in PB, but conversely increased in BM. Notably, FTY720 treatment prevented the development of colitis that is otherwise induced in untreated SPX LT‐α^?/?×RAG‐2^?/? mice upon transfer of colitic lamina propria CD4⁺ T cells. In such mice, the number of CD4⁺ T cells in PB or lamina propria of FTY720‐treated SPX LT‐α^?/?×RAG‐2^?/? recipients was significantly reduced, but that in the BM was significantly increased as compared with untreated control mice. Altogether, the present results indicate that FTY720 treatment may offer an additional role to direct trafficking of CD4⁺ T cells in BM, resulting in the prevention of colitis.     

CD38+ CD45RBlow CD4+ T cells: a population of T cells with immune regulatory activities in vitro

An antibody reactive with CD38 revealed both phenotypic and functional heterogeneity amongst CD45RB^low cells. Functional analysis of the CD38⁺ and CD38⁻ fractions showed that the latter contained T cells which responded to recall antigens and produced high levels of cytokine in response to polyclonal stimulation. In contrast, the CD38⁺ population failed to proliferate or to produce detectable levels of cytokines. Despite appearing unresponsive, the CD38⁺ population significantly inhibited anti-CD3-induced proliferation and cytokine secretion by the reciprocal CD38⁻ population. Immune suppression required stimulation through the TCR and was dependent on a physical interaction between regulatory and responding CD4⁺ populations. It did not involve killing of the responding T cells or secretion of IL-10 or TGF-β. Despite some similarities there is no direct correlation between the in vitro suppression characteristic of the CD38⁺ CD45RB^low subset and in vivo suppression which has been shown to be mediated by unseparated CD45RB^low CD4⁺ T cells. However, these results demonstrate that two functionally distinct subsets of T cells reside within the antigen-exposed or CD45RB^low CD4⁺ T cell population and are thus generated in vivo: (1) conventional memory T cells which proliferate and secrete cytokines in response to activation and (2) a population of regulatory T cells which inhibit T cell activation in vitro. Antibodies reactive with CD38 may provide a useful tool with which to study the role of these T cell subsets in the induction and regulation of the immune response.     

Immunoglobulin leakiness in scid mice with CD4(+) T-cell-induced chronic colitis

Inflammatory bowel disease in scid mice is initiated by transplantation of CD4(+) T-cells from immunocompetent syngenic donor mice. As the disease progresses, immunoglobulin (Ig)-containing cells appear in the gut lamina propria, suggesting that locally accumulating Ig may play a role in disease development. In the present work we have investigated the relationship between disease progression and patterns or levels of Ig isotypes in the feces of scid mice suffering from an ongoing colitis. The data clearly showed that the severity or progression of the disease did not influence the levels of IgA, IgG1, IgG2a, IgG2b, and IgG3, whereas the level of fecal IgM increased during the course of colitis. The presence of the serum protein alpha-1-antitrypsin in fecal extracts from diseased mice suggests that some of the fecal Ig has leaked through the inflamed epithelial membrane into the gut lumen. Finally, Ig-containing cells were observed in mesenteric lymph nodes and in the spleen, suggesting that the fecal Ig is produced both systemically and locally in the gut wall. In conclusion, the present results demonstrate that the level of IgM increases as colitis progresses. Also, the five remaining major Ig isotypes are increased in the gut lumen of scid mice with colitis, but the individual Ig types vary randomly during the course of the disease. Thus, it is unlikely that immunoglobulins are involved in the immunopathogenesis of this model of colitis.     

Evidence that CD4+, but not CD8+ T cells are responsible for murine interleukin-2-deficient colitis

Mice deficient in interleukin-2 production (IL-2^null mice) develop colonic inflammation closely resembling ulcerative colitis in humans. Although this disease is marked by substantial infiltration of the colon by CD8⁺ and CD4⁺ T lymphocytes, no function has yet been assigned to these T cell subsets in the development of colitis in the IL-2^null mouse. For the present study, we investigated the involvement of T lymphocytes in the onset of colitis in IL-2^null mice, and examined the possible role played by cytotoxic T cells. Both lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) of the colon of IL-2^null mice were potently cytotoxic ex vivo in short-term redirected cytotoxic lymphocyte (CTL) assays. In contrast, colonic T cells of wild-type animals showed little or no constitutive cytotoxic T cell activity. Colonic CTL were detectable prior to the appearance of disease in IL-2^null animals and CTL activity was confined to the TcRαβ, rather than to the TcRγδ IEL subset. IL-2^null animals crossed with major histocompatibility complex class I-deficient mice [IL-2^null × β₂ microglobulin (β₂m^null] mice also developed colitis, which appeared even earlier than in most IL-2^null mice. These findings suggest that neither CD8⁺ IEL nor LPL were causal in the onset of colitis in IL-2^null animals. In IL-2^null × β₂m^null mice, an ulcerative colitis-like disease was evident from histological studies and immunohistological staining which showed very large numbers of CD4⁺ lymphocytes within the intestinal mucosa. Significant ex vivo killing by CD4⁺ T cells was observed in IL-2^null × β₂m^null animals, although this required an extended incubation time compared to colonic CD8⁺ T cells. Peripheral as well as colonic CD4⁺ T cells in IL-2^null and IL-2^null × β₂m^null animals, were activated as judged by their cell surface phenotype (CD45RB^lo, L-selectin^lo and CD69⁺). In light of these findings, we propose that infiltrating CD4⁺, but not CD8⁺ T cells are central to the inflammation observed in the intestinal mucosa in IL-2^null colitis.     

Immunosenescent colitogenic CD4(+) T cells convert to regulatory cells and suppress colitis

Inflammatory bowel diseases progress steadily by the expansion of colitogenic CD4(+) cells. However, it remains unknown whether colitogenic CD4(+) cells are long-living like memory cells or exhausted like effector cells. To assess the longevity of colitogenic lamina propria (LP) CD4(+) cells, we performed sequential transfers of LP CD4(+) cells from colitic CD4(+)CD45RB(high) cell-transferred SCID mice into new SCID mice. Although SCID mice transferred with colitic LP CD4(+) cells stably developed colitis until at least the sixth transfer, the interval to the development of colitis gradually lengthened as the number of transfers increased. The incidence of colitis gradually decreased after the seventh transfer. Furthermore, non-colitic LP CD4(+) cells from mice transferred over seven times expressed significantly higher levels of PD-1 and produced significantly lower amounts of IFN-gamma, TNF-alpha, and IL-17 than colitic LP CD4(+) cells recovered after the first transfer. Most notably, we found that re-transfer of non-colitic LP CD4(+) cells recovered after multiple transfers prevented the development of colitis in SCID mice co-transferred with CD4(+)CD45RB(high) cells. Thus, colitogenic LP CD4(+) cells may be exhausted over time, become non-functional, convert to regulatory cells, and finally suppress colitis in the process of immunosenescence.     

A role for CD4+ T cells in the pathogenesis of skin fibrosis in tight skin mice

The tight skin (Tsk/+) mouse represents a murine model of heritable fibrosis with some similarities to the skin fibrosis seen in human scleroderma. Tsk/+ animals display alterations in connective tissue in some internal organs. Skin fibrosis can be adoptively transferred to normal recipients with Tsk/+ bone marrow or spleen cells and older Tsk/+ animals develop autoantibodies against topoisomerase suggesting that some of the pathogenesis in the Tsk/+ mouse may be mediated by autoimmunity. To determine the role of T cell subsets in the pathogenesis of fibrotic disease, Tsk/+ mice were bred with CD4- and CD8-deficient (CD4^−/− and CD8^−/−) mice. Tsk/+ CD4^−/− mice showed a marked reduction in skin fibrosis as well as decreased cellularity and only mild collagen disorganization as compared to Tsk/+ CD4⁺ CD8⁺ control mice yet did not differ from Tsk controls in the level of serum anti-topoisomerase activity. In contrast, Tsk/+ CD8^−/− mice exhibited the same histology in the skin as Tsk/+ controls yet had significantly reduced levels of serum anti-topoisomerase activity. Lung pathology, i.e. emphysema, was unaffected by both the CD4 or CD8 mutations. These data show that only some of the pathological effects of the Tsk mutation are T cell dependent and that different T cell subsets affect different parameters in this multi-organ model of fibrotic disease.     

Circulating CD4+CD8+ T lymphocytes in patients with Kawasaki disease

We serially monitored cell surface antigen expression on mononuclear cells in peripheral blood isolated from patients with Kawasaki disease (KD), and found, for the first time, that a markedly increased number of CD4⁺CD8⁺ T lymphocytes was present in some of the patients (11 of the 24 cases). The cases of five of these 11 patients were complicated with coronary artery lesion (CAL); the 13 patients with normal numbers of CD4⁺CD8⁺ T lymphocytes did not have CAL. The patients' age, sex and grade of systemic inflammation evaluated by peripheral leucocyte count and serum C-reactive protein levels were not correlated to the number of CD4⁺CD8⁺ T lymphocytes. Other cell surface antigen characteristics of the CD4⁺CD8⁺ T lymphocytes included CD3⁺, CD45RA⁺, CD45RO⁺, CD16^?, and HLA-DR⁺. These results indicate that the surface antigen characteristics of the KD peripheral blood examined were the same as those of Epstein–Barr virus infection without CD45RA⁺. These findings provide useful information for the analysis of the pathogenesis of KD.     

小鼠CD4＋ CD25＋免疫调节性T细胞体外扩增及功能检测的研究

目的 探讨小鼠CD4＋ CD25＋调节性T细胞（Tregs）的体外扩增方法及扩增后Tregs的免疫功能.方法 利用免疫磁珠法分离小鼠Tregs;采用anti-CD3mAb＋rIL-2和Allo-APC＋rIL-2两种方法进行体外扩增,通过TRANSWELL培养系统、细胞因子表达及CFSE染色标记等方法检测Tregs免疫抑制作用的机制.结果 体外分离的Tregs纯度为89.5％;活细胞率约为98％.Tregs扩增的倍数在两组各时间点差异无统计学意义.扩增后CD4＋ CD25＋T细胞、CD4＋T细胞和CD4＋ CD25-T细胞的每分钟计数（CPM）分别为1 470、12 700和14 300.混合淋巴细胞反应（MLR）中当CD4＋：CD25＋T细胞的比例为1∶1时,抑制率为79％,比例为8∶1时,抑制率为33％.CFSE标记后,进入分裂周期的CD4＋T细胞的百分比在未加入CD4＋ CD25＋T细胞组为83.7％,加入CD4＋ CD25＋T细胞组为31.7％,差异具有统计学意义（P=0.0006）.CD4＋ CD25＋T细胞对CD4＋T细胞的增殖抑制率在Transwell和Non-Transwell的培养系统中分别为＜5％和95％.Transwell培养组中IL-2含量高于Non-Transwell组,差异具有统计学意义[Transwell培养组为（158.33±2.08）pg/mL,Non-Transwell组为（23.00±2.00） pg/mL,P ＜0.0001].结论 采用免疫磁珠法分离小鼠Tregs可行,Tregs可有效扩增;Tregs的作用机制为抑制CD4＋T细胞的增殖及抑制CD4＋T细胞分泌IL-2;其抑制作用需要细胞-细胞间接触;体外扩增后的Tregs仍具有免疫特性,其体外免疫抑制作用增强.     

CD4+ CD25+调节性T细胞在流产中的研究进展

CD4+CD25+调节性T细胞(Tr)是一个具有独特免疫调节功能的T细胞亚群.Tr免疫学特性主要在于抑制自身反应性T细胞的活化,并参与外周免疫耐受,对维持机体内环境的稳定起重要作用.Tr在流产中发挥重要的免疫调节作用.     

天然CD4+CD25+Treg细胞的研究进展

天然CD4+ CD25+ Treg细胞在针对自身抗原和外来抗原的免疫应答中起关键控制作用,其缺乏或功能性的缺陷将导致多重病理性的失调.本文就近年在其产生、作用机制以及与免疫耐受的诱导关系等方面的研究进展进行了综述.     

Single-cell analyses of CD4+ T cells from αβ T cell receptor-transgenic mice: a distinct mucosal cytokine phenotype in the absence of transgene-specific antigen

Development of distinct CD4⁺ T cell cytokine phenotypes may be conditioned by the anatomic site in which activation occurs. A double-label in situ hybridization technique was used to characterize co-expression of cytokine mRNA in antigen-specific responses of Peyer's patch (PP), lamina propria (LP), and splenic (SP) CD4⁺ T cells isolated from αβ T cell receptor-transgenic mice. Interleukin (IL)-2 was the dominant cytokine expressed by antigen-stimulated PP and SP populations, though it was expressed by a minority of the activated T cells. Cells that expressed interferon (IFN)-γ were less frequent, and IL-4, IL-5, and IL-10 were infrequent. In contrast, cells that expressed IFN-γ or IL-10 were most frequent in the LP population, with lower frequencies of IL-2, and few IL-4- and IL-5-positive cells. Co-expression of two cytokines by the same cell was the exception, regardless of the anatomic site from which the T cells were isolated. The surface phenotype of transgene-positive T cells isolated from each anatomic site was distinct, despite the absence of in vivo exposure to antigen for which the transgenic T cell receptor is specific. These data suggest that the cytokine responses of CD4⁺ T cells may be conditioned by the microenvironment, independently of specific antigen, and that the LP CD4⁺ T population has a distinct cytokine expression pattern with counter-regulatory properties that may be important for homeostasis in mucosal immune tissues.     

MHC class II-independent CD25+ CD4+ CD8alpha beta+ alpha beta T cells attenuate CD4+ T cell-induced transfer colitis

CD4+ alpha beta T cell populations that develop in mice deficient in MHC class II (through 'knockout' of either the Aalpha, or the Abeta chain of the I-A(b) molecule) comprise a major 'single-positive' (SP) CD4+ CD8- subset (60-90%) and a minor 'double-positive' (DP) CD4+ CD8alpha beta+ subset (10-40%). Many DP T cells found in spleen, mesenteric lymph nodes (MLN) and colonic lamina propria (cLP) express CD25, CD103 and Foxp3. Adoptive transfer of SP but not DP T cells from Aalpha(-/-) or Abeta(-/-) B6 mice into congenic RAG(-/-) hosts induces colitis. Transfer of SP T cells repopulates the host with only SP T cells; transfer of DP T cells repopulates the host with DP and SP T cells. Anti-CD25 antibody treatment of mice transplanted with DP T cells induces severe, lethal colitis; anti-CD25 antibody treatment of mice transplanted with SP T cells further aggravates the course of severe colitis. Hence, regulatory CD25+ T cells within (or developing from) the DP T cell population of MHC class II-deficient mice control the colitogenic potential of CD25- CD4+ T cells.