Downregulation of CD4＋CD25＋ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.     

Gram-negative bacteria and phagocytic cell interaction mediated by complement receptor 3

Complement receptor 3 (CR3) is an integrin that recognizes several different ligands. Binding to CR3 in phagocytic cells activates signaling pathways involved in cytoskeleton rearrangement, regulation of cell motility, alteration of gene expression and phagocytosis of complement-opsonized as well as of some non-opsonized particles and pathogenic bacteria. However, CR3-mediated phagocytosis of some Gram-negative bacteria does not induce bacterial clearance. Pseudomonas aeruginosa, Salmonella and Escherichia coli are eliminated after phagocytic cell-bacteria interaction mediated by CR3. However, Bordetella takes advantage of the CR3 function and uses it to enter into macrophages leading to bacterial survival. The final fate of the pathogen is determined by combinations of host and bacterial factors, in which molecular interactions between CR3 and bacterial ligands are involved.     

Expression of complement regulatory proteins [membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59)] in endometrial stressed cells

In the female reproductive tract, the complement system represents a defense mechanism that can act directly against pathogens and cells, and mediates inflammatory response. Endometrial cells are protected from autologous complement attack by membrane-bound complement regulatory proteins (CRPs) that prevent complement activation: membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59). In this work we show that all CRPs were overexpressed after LPS exposure. Maximal stimulatory effect was detected after 6h, and was declining after 12h, reaching control levels in 24h. CD59 was the protein showing the more prominent effect. There seems to be a slight increase of CRP expression in the endometrium of sterile patients that have anti-endometrial antibodies (AEA) in their serum. Our results suggest that under stress, the high expression of CRPs (CD46, CD55, and CD59) could protect endometrial injured cells against complement mediated lysis. The survival of these cells with some biochemical modifications would enable autoimmune response.     

Induction of FoxP3+CD4+CD25+ regulatory T cells by a bone marrow population distinct from plasmacytoid-DC

Facilitating cells (FC) are bone marrow-derived cells that facilitate allogeneic hematopoietic stem cell (SC) engraftment and induce transplantation tolerance without causing graft vs. host disease. Although there is evidence for FC directing the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells, the specific FC subsets that control regulatory T cell development have not been defined. The current study investigates the role of FC-CD3ε⁺ and FC-CD3ε⁻ subpopulations in the development of FoxP3⁺CD4⁺CD25⁺ regulatory T cells. Here, we demonstrate that the induction of FoxP3⁺CD4⁺CD25⁺ regulatory T cells in coculture is mediated by not only the FC-CD3ε⁻ subset but also the FC-CD3ε⁺ subset, which is distinct from plasmacytoid precursor dendritic cells (p-preDC). The identification of cell populations distinct from p-preDC that efficiently induce the generation of FoxP3⁺CD4⁺CD25⁺ regulatory T cells may prove useful for future therapeutic applications for the induction of tolerance following allogeneic SC transplantation.     

CD4+CD25+ regulatory T cells control innate immune reactivity after injury

Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1(-/-)) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-alpha, IL-1beta, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1(-/-), CD4(-/-), or CD8(-/-) mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8(-/-) mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4(-/-) mice produced cytokines at significantly higher levels, equivalent to those in Rag1(-/-) mice. Moreover, reconstitution of Rag1(-/-) or CD4(-/-) mice with WT CD4(+) T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4(+) T cells into CD4(+)CD25(+) and CD4(+)CD25(-) subpopulations before their adoptive transfer into Rag1(-/-) mice showed that CD4(+)CD25(+) T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4(+)CD25(-) T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4(+)CD25(+) T regulatory cells in controlling host inflammatory responses after injury.     

CD4+CD25+ regulatory T cells control the progression from periinsulitis to destructive insulitis in murine autoimmune diabetes

Non-obese diabetic (NOD) mice develop spontaneous T-cell responses against pancreatic beta-cells, leading to islet cell destruction and diabetes. Despite high genetic similarity, non-obese resistant (NOR) mice do not develop diabetes. We show here that spleen cells of both NOD and NOR mice respond to the islet cell antigen glutamic acid decarboxylase-65 in IFN-gamma-ELISPOT assays. Moreover, NOR-T cells induce periinsulitis in NOD SCID recipient mice. Thus, a potentially pathogenic islet cell-specific T-cell response arises in NOR and NOD mice alike; the mechanism that prevents the autoimmune progression of self-reactive T cells in NOR mice presumably acts at the level of effector function. Consistent with this hypothesis, CD4+CD25+ cell-depleted spleen cells from NOR mice mediated islet cell destruction and overt diabetes in NOD SCID mice. Therefore, islet cell-specific effector cells in NOR mice appear to be under the control of CD4+CD25+ regulatory T cells, confirming the importance of regulatory cells in the control of autoimmune diabetes.     

Novel role of aquaporin-4 in CD4+ CD25+ T regulatory cell development and severity of Parkinson's disease

Aquaporin-4 (AQP4) is highly expressed in mammalian brains and is involved in the pathophysiology of cerebral disorders, including stroke, tumors, infections, hydrocephalus, epilepsy, and traumatic brain injury. We found that AQP4-deficient mice were hypersensitive to stimulations such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or lipopolysaccharide compared to wild-type (WT) littermates. In a mouse model of MPTP-induced Parkinson's disease (PD), AQP4-deficient animals show more robust microglial inflammatory responses and more severe loss of dopaminergic neurons (DNs) compared with WT mice. However, a few studies have investigated the association of abnormal AQP4 levels with immune dysfunction. Here, for the first time, we report AQP4 expression in mouse thymus, spleen, and lymph nodes. Furthermore, the significantly lower numbers of CD4(+) CD25(+) regulatory T cells in AQP4-deficient mice compared to WT mice, perhaps resulting from impaired thymic generation, may be responsible for the uncontrolled microglial inflammatory responses and subsequent severe loss of DNs in the substantia nigra pars compacta in the MPTP-induced PD model. These novel findings suggest that AQP4 deficiency may disrupt immunosuppressive regulators, resulting in hyperactive immune responses and potentially contributing to the increased severity of PD or other immune-associated diseases.     

CD4+CD25+调节性T细胞在自身免疫性肝炎发病中的作用

目的:比较自身免疫性肝炎(autoimmune hepatitis,AIH)患者与健康对照者(healthy controls,HCs)外周血CD4+CD25+调节性T细胞(CD4+CD25+Tregs)数量、免疫抑制功能的变化,探讨CD4+CD25+Tregs参与AIH发病的可能机制.方法:采用流式细胞仪检测8例AIH患者及15例健康对照组的外周血CD4+CD25+Tregs数量的百分比及绝时数量;采用共同培养方法检测AIH患者外周血CD4+CD25+Tregs的免疫抑制功能的变化;实时荧光定量聚合酶链反应(RT-FQ-PCR)检删AIH患者外周血CD4+CD25+Tregs中FoxP3mRNA的表达.结果:AIH患者外周血CD4+CD25+Tregs数量明显低于HCs(p<0.01);混合淋巴细胞共同培养结果显示,AIH患者外周血CD4+CD25+Tregs抑制功能明显低于HCs组(p<0.01);AIH患者外周血CD4+CD25+Tregs的FoxP3 mRNA相对表达量显著降低,与HCs组比较有显著性差异(p<0.01).结论:CD4+CD25+Tregs细胞的数量的减少和Foxp3表达的降低所造成的CD4+CD25+Tregs细胞免疫抑制功能受损可能是AIH发病的一个因素.     

Follicular lymphoma intratumoral CD4+CD25+GITR+ regulatory T cells potently suppress CD3/CD28-costimulated autologous and allogeneic CD8+CD25- and CD4+CD25- T cells

Regulatory T cells (T(R)) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ T(R) that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These T(R) actively suppress both the proliferation of autologous nodal CD8+CD25- and CD4+CD25- T cells, as well as cytokine production (IFN-gamma, TNF-alpha and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these T(R) are also capable of suppressing the proliferation of allogeneic CD8+CD25- and CD4+CD25- T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25- and CD4+CD25- T cells, suggesting that FLN T(R) are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-beta signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-beta in FLN T(R)-mediated suppression.     

Impairment of regulatory capacity of CD4+CD25+ regulatory T cells mediated by dendritic cell polarization and hyperthyroidism in Graves' disease

Graves' disease (GD) is one of the most common autoimmune diseases. The immune dysfunction in GD involves the generation of thyroid-stimulating hormone receptor (TSHR) autoantibodies that presumably arise consequent to interactions among dendritic cells (DCs), T cells, and regulatory T (Treg) cells. However, the immunological mechanisms of interactions between them that lead to the induction and regulation of this autoimmune disease are poorly defined. In this study, we investigated whether DCs are the main cause of the defective activity of Treg cells in GD patients. We found a significant decrease in the percentage of circulating CD4(+)CD25(+)FOXP3(+) Treg cells in untreated GD patients (uGD), which was negatively correlated with the concentration of TSHR autoantibodies. uGD-derived DCs were polarized to increase the number of plasmacytoid DCs (pDCs) and conferred the ability to abrogate the suppressive function of Treg cells through inducing apoptosis of CD4(+)CD25(+) Treg cells in an IFN-α-dependent manner, and elevated thyroid hormones further exacerbated the effect. The nucleotide UDP, which inhibits IFN-α secretion of pDCs through P2Y6 receptor signaling, restored the suppressive function of CD4(+)CD25(+) Treg cells. Collectively, uGD-derived DCs through pDC polarization and elevated thyroid hormones act in concert to impair the regulatory capacity of Treg cells, facilitating the production of TSHR autoantibodies in the pathogenesis of GD.     

牙龈卟啉单胞菌脂多糖对CD4^＋CD25^＋调节性T细胞免疫抑制功能的影响

目的探讨牙龈卟啉单胞菌（Porphyromonas gingivalis,Pg）对CD4^＋CD25^＋调节性T细胞（regulatory T cells,Tregs）免疫抑制功能的影响。方法采用酚水法提取Pg ATCC 33277株脂多糖（lipopolysaccharide,LPS）。免疫磁珠法分离BALB／c小鼠脾脏CD4^＋CD25^＋Tregs并进行体外培养,同时给予不同剂量（0～500ng／ml）Pg—LPS干预,培养48h后收集细胞及上清液。Real-TimePCR法测定培养细胞Foxp3mRNA的表达,ELISA法分别测定细胞上清液中IL-10、TGF-β水平;采用体外淋巴细胞混合培养法对Pg-LPS干预后的CD4^＋CD25^＋Tregs进行功能抑制试验。结果Pg-LPS干预不影响CD4^＋CD25^＋Tregs分泌IL-10和TGF-β,但是能够显著上调CD4^＋CD25^＋TregsFoxp3mRNA的表达,增强其免疫抑制作用;当Ps—LPS浓度低于300ng／m1时,CD4^＋CD25^＋TregsFoxp3mRNA表达以及免疫抑制作用的增强与Ps—LPS浓度之间呈剂量-效应关系。结论Pg-LPS能够增强CD4^＋CD25^＋Tregs的免疫抑制作用,这种免疫抑制增强效应可能与CD4^＋CD25^＋Tregs Foxp3基因表达的上调有关,并且不具有抑制性细胞因子依赖性。     

Immune regulation and control of regulatory T cells by OX40 and 4-1BB

The TNFR family members OX40 (CD134) and 4-1BB (CD137) have been found to play major roles as costimulatory receptors for both CD4 and CD8 T cells. In particular, in many situations, they can control proliferation, survival, and cytokine production, and hence are thought to dictate accumulation of protective T cells during anti-viral and anti-tumor responses and pathogenic T cells during autoimmune reactions. As opposed to simply controlling the activity of naïve, effector, and memory T cells, recent data have suggested that both molecules are also instrumental in controlling the generation and activity of so-called regulatory or suppressor T cells (Treg), perhaps in both positive and negative manners. Part of the action on Treg might function to further promote protective or pathogenic T cells, but alternate activities of OX40 and 4-1BB on Treg are also being described that suggest that there might be control by these molecules at multiple levels that will alter the biological outcome when these receptors are ligated. This review specifically focuses on recent studies of regulatory T cells, and regulatory or suppressive activity, that are modulated by OX40 or 4-1BB.     

Paclitaxel may inhibit autoimmune diseases by sparing or increasing the number of CD4(+) CD25(+) regulatory T cells

Paclitaxel, a representative of taxanes, exhibits cytotoxic effects against a broad range of tumors. Strikingly, an emerging body of data suggests that paclitaxel also exerts effects on immune system by stimulating anti-tumor and anti-autoimmunity effects, supporting the idea that paclitaxel suppresses tumor through several mechanisms and not solely through inhibiting cell division. Based on the accumulating data, we hypothesized that paclitaxel may inhibit autoimmune diseases by sparing or actively increasing the number of CD4(+) CD25(+) Treg cells. The hypothesis, if proved to be correct, will significantly improve our understanding of the tumor immunity, autoimmunity and its related pathological effects. It will influence our choice on immunosuppressive drugs for cancer patients with autoimmune diseases. It will also impact the immunotherapy for tumors.     

Activation of CD25(+)CD4(+) regulatory T cells by oral antigen administration

CD25(+)CD4(+) T cells are naturally occurring regulatory T cells that are anergic and have suppressive properties. Although they can be isolated from the spleens of normal mice, there are limited studies on how they can be activated or expanded in vivo. We found that oral administration of OVA to OVA TCR transgenic mice resulted in a modification of the ratio of CD25(+)CD4(+) to CD25(-)CD4(+) cells with an increase of CD25(+)CD4(+) T cells accompanied by a decrease of CD25(-)CD4(+) T cells. The relative increase in CD25(+)CD4(+) T cells persisted for as long as 4 wk post feeding. We also found that CTLA-4 was dominantly expressed in CD25(+)CD4(+) T cells and there was an increase in the percentage of CD25(+)CD4(+) T cells expressing CTLA-4 in OVA-fed mice. In contrast to CD25(-)CD4(+) cells, CD25(+)CD4(+) cells from fed mice proliferated only minimally to OVA or anti-CD3 and secreted IL-10 and elevated levels of TGF-beta(1) following anti-CD3 stimulation. CD25(+)CD4(+) cells from fed mice suppressed the proliferation of CD25(-)CD4(+) T cells in vitro more potently than CD25(+)CD4(+) T cells isolated from unfed mice, and this suppression was partially reversible by IL-10 soluble receptor or TGF-beta soluble receptor and high concentration of anti-CTLA-4. With anti-CD3 stimulation, CD25(+)CD4(+) cells from unfed mice secreted IFN-gamma, whereas CD25(+)CD4(+) cells from fed mice did not. Adoptive transfer of CD25(+)CD4(+) T cells from fed mice suppressed in vivo delayed-type hypersensitivity responses in BALB/c mice. These results demonstrate an Ag-specific in vivo method to activate CD25(+)CD4(+) regulatory T cells and suggest that they may be involved in oral tolerance.