CD4+CD25+ regulatory T lymphocytes in bone marrow transplantation

Induction of immunological tolerance to alloantigens would be the treatment of choice to prevent graft-versus-host disease (GvHD) and allograft rejection in transplantation medicine. Organisms use a variety of mechanisms to avoid potentially deadly immunity to self-antigens. The most potent self-tolerance mechanism is probably dominant tolerance assured by regulatory and suppressor T lymphocytes. It appears therefore attractive to use the same mechanism to induce transplantation-tolerance. We here review and discuss recent advances in the use of one of the best-characterized regulatory T lymphocyte populations, CD4(+)CD25(+) T cells, to prevent graft-versus-host disease and bone marrow allograft rejection.     

Human mast cells present antigen to autologous CD4+ T cells

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Regulation of allergen-induced bone marrow eosinophilopoiesis: role of CD4+ and CD8+ T cells

BACKGROUND: The mechanisms of the distant stimulation of the bone marrow (BM) after airway allergen exposure remain largely obscure. T cells have been implicated in allergic airway inflammation but their role in allergen-induced BM eosinophilopoiesis is poorly understood. The aim of this study was to determine the role of CD4(+) and CD8(+) T cells in allergen-induced BM eosinophilopoiesis. METHODS: Ovalbumin (OVA)-sensitized wild type (WT), CD4 knockout (CD4-/-) and CD8 knockout (CD8-/-) mice were exposed intranasally to OVA or saline. Bromo-deoxyuridine (BrdU) was used to label newly produced cells. Bone marrow, blood and bronchoalveolar lavage (BAL) were sampled 24 h after the final exposure. Immunostaining for newly produced eosinophils (i.e. BrdU(+)/MBP(+)) and BM eosinophil progenitor [CD34(+)/CD45(+)/interleukin-5 (IL-5)Ralpha(+)] cells was performed. RESULTS: The number of newly produced BM eosinophils (BrdU(+)/MBP(+) cells) was significantly reduced in allergen exposed CD4-/- or CD8-/- mice compared with allergen exposed WT mice, which was followed by a subsequent decrease in newly produced blood and airway eosinophils. Furthermore, BM eosinophil progenitors were significantly reduced in allergen exposed CD4-/- and CD8-/- mice compared with WT mice. Finally, serum IL-5 and Bronchoalveolar lavage fluid eotaxin-2 levels were abolished in allergen exposed CD4-/- mice to levels seen in saline exposed WT mice. CONCLUSIONS: These data suggests that both CD4(+) and CD8(+) T cells have a regulatory role in allergen-induced BM eosinophilopoiesis, whereas CD4(+) T cells are obligatory for allergen-induced airway eosinophilia. The subsequent traffic of eosinophils to the airways is likely to be at least partly regulated by a CD4(+) T-cell-dependent local airway eotaxin-2 production.     

骨髓间充质干细胞对肾病综合征大鼠CD4+CD25+调节性T细胞的影响

背景：CD4⁺CD25⁺调节性T细胞功能降低、数量下降已被公认为是肾病综合征患儿免疫失调的重要表现。骨髓间充质干细胞具有免疫调节功能,可上调CD4⁺CD25⁺调节性T细胞,抑制淋巴细胞增殖,并已在许多免疫性疾病方面成功应用。 目的：观察骨髓间充质干细胞移植对原发性肾病综合征大鼠外周血CD4⁺CD25⁺调节性T细胞的影响。 方法：自SD大鼠分离骨髓间充质干细胞,经体外传代培养及鉴定后制备细胞悬液。30只SD大鼠随机均分为3组,生理盐水组和干细胞移植组尾静脉注射阿霉素建立阿霉素肾病大鼠模型,干细胞移植组注射阿霉素当日尾静脉射干细胞1×10⁷;正常组不做处理。 结果与结论：①与正常组比较,生理盐水组大鼠均出现肾病综合征表现,以腹水、大量蛋白尿、低蛋白血症、高胆固醇血症为特征;干细胞移植组较生理盐水移植组则有明显改善(P < 0.05)。②造模第28天,干细胞移植组和生理盐水组大鼠造模后外周血CD4⁺CD25⁺Treg/ CD4⁺ Treg均明显高于正常组(P < 0.05);干细胞移植组与生理盐水组比较差异无显著性意义(P > 0.05)。③造模第28天,干细胞移植组大鼠外周血单个核细胞FoxP3mRNA的表达显著高于生理盐水组和正常组(P < 0.05)。提示骨髓间充质干细胞移植对阿霉素肾病综合征模型有一定的治疗作用,其机制可能与上调FoxP3在组织局部的表达而诱导CD4⁺CD25⁺Treg产生有关。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Resident CD8+ T cells suppress CD4+ T cell-dependent late allergic airway responses

BACKGROUND: The role of CD8+ T cells in the immune response to airway challenge with an allergen is poorly understood. OBJECTIVE: The aim of this study was to test the hypothesis that resident naive CD8+ T cells modulate the magnitude of CD4+ T cell-dependent allergic airway responses. METHODS: Cervical lymph node CD4+ T cells (2 x 10(6)) were harvested from ovalbumin (OVA)- or sham-sensitized rats and injected intraperitoneally into naive Brown Norway recipients. The recipients were treated with a CD8alpha mAb (OX-8) to deplete the resident CD8+ T cells (n = 12) or mouse ascites (n = 12). Two days after adoptive transfer, the recipient animals were OVA challenged, lung resistance was measured for 8 hours, and bronchoalveolar lavage (BAL) was performed. RESULTS: After OVA challenge, primed CD4-transferred CD8-depleted rats had larger early airway responses and late airway responses compared with primed CD4-transferred CD8-nondepleted rats (early airway responses: 158.6% +/- 19.2% vs 115.7% +/- 5.9%, P < .05; late airway responses: 8.5% +/- 1.7% vs 4.4% +/- 0.9%, P < .05). BAL eosinophilia was also greater (4.67% +/- 0.45% vs 2.34 +/- 0.26%, P < .01). The cells in BAL fluid expressing IL-4 mRNA were not significantly changed by CD8 depletion, but IL-5 mRNA+ cells were higher in number, and IFN-gamma mRNA+ cells were fewer in the CD8-depleted group. CONCLUSIONS: Resident CD8+ T cells downregulate the late allergic response and airway inflammation evoked by CD4+ T-cell transfers in Brown Norway rats. This downregulation does not require antigen priming.     

胸腺基质细胞支持下骨髓CD34~+细胞向T细胞体外定向分化

目的：研究人骨髓CD34^＋细胞体外向T细胞定向分化的方法，为研究造血细胞淋系造血活性及T细胞发育和分化提供技术平台。方法：免疫磁珠法分离骨髓CD34^＋细胞，在骨髓基质细胞条件培养液构建的微环境下，在胸腺基质细胞的支持下，使其体外向T细胞定向分化，收集培养的非贴壁细胞，免疫荧光染色后经流式细胞术检测培养不同时间CD1^-CD3^＋细胞、CD3^＋CD4^＋CD8^-细胞及CD3^＋CD4^-CD8^＋细胞比例。结果：培养1周时，培养细胞中以不成熟的CD1^＋CD3^-细胞、CD1^＋CD3^＋细胞为主，可检测到少量CD1^-CD3^＋细胞，随培养时间延长，不成熟细胞比例逐渐减少，而成熟的CD1^-CD3^＋细胞比例逐渐增加；在CD3^＋细胞中，培养初期以不成熟的双阳性细胞CD4^＋CD8^＋为主，而成熟的单阳性CD^＋CD8^-细胞及CD4^-CD8^＋细胞占极小比例，随培养时间延长，双阳性细胞比例逐渐减少，而成熟的单阳性细胞比例逐渐增高；而无胸腺基质细胞支持的CD34＋细胞仅在培养初期检测到成熟细胞存在，而培养后4周基本检测不到成熟T细胞的存在。结论：在骨髓基质细胞及胸腺基质细胞的支持下，骨髓CD34^＋细胞可体外发育为成熟的CD1^-CD3^＋细胞及单阳性T细胞，其中胸腺基质细胞的支持对于造血细胞向T细胞的体外定向分化极其重要。     

Depletion of CD34+ CD4+ cells in bone marrow from HIV-1-infected individuals.

Pancytopenia as a consequence of bone marrow abnormalities is commonly seen in HIV-infected individuals. To examine the effect that HIV-1 has on hematopoietic cells, we compared hematopoietic properties of bone marrow samples from HTV+ patients at various stages of disease with bone marrow samples from uninfected donors. While the absolute number of recovered CD34+ cells and the cloning efficiency of these cells did not differ significantly in HIV+ donors, the percentage of CD34+ CD4+ cells was significantly depleted in late-stage HIV+ patients. We observed a direct correlation between the numbers of CD34+ CD4+ cells in the bone marrow and the peripheral CD4 count. Further characterization of the CD34+ CD4+ subpopulation demonstrated that these cells expressed lower levels of HLA-DR on their surface compared with CD34+ CD4- cells, suggesting an immature phenotype. We also found evidence for expression of HIV-1 coreceptors CXCR-4 and CKR-5 message and protein in CD34+ bone marrow cells. While this finding suggested that hematopoietic cells might be susceptible to HIV infection at an early stage of maturation, thus affecting different cell lineages as they matured, we did not find any evidence for infection of HIV in these cells. These data suggest that HIV affects early hematopoietic progenitor cells either directly or indirectly, and in particular CD34+ CD4+ cells. This finding has important implications for disease pathogenesis and for application of gene therapy approaches that use CD34+ hematopoietic cells.     

CD4+CD25+-regulatory T cells from mouse to man

Immunological tolerance is one of the fundamental concepts of the immune system. During the past decade, CD4+CD25+-regulatory T cells have emerged as key players in the development of tolerance to autoantigens as well as to foreign antigens. Still many questions remain illusive regarding the basic properties of CD4+CD25+-regulatory T cells. This review aims to recapitulate some of the current understandings about the phenotype, function and clinical relevance of murine and human CD4+CD25+-regulatory T cells.     

CD4+CD45RA+T cells from adults respond to recall antigens after CD28 ligation

The leukocyte common antigen isoforms CD45RA and CD45RO havelong been used to discriminate human naive and memory T cellsrespectively. This model was largely based on the observationthat CD45RO⁺ T cells respond preferentially to and show a higherfrequency of precursors specific for recall antigens. However,CD45RA⁺ T cells have more stringent requirements for stimulationand standard in vitro assays may favour CD45RO⁺ cells in thisrespect. We tested the hypothesis that CD45RAf T cells respondpoorly to in vitro stimulation with recall antigens becauseof inadequate stimulation rather than a lack of precursors.Limiting dilution analyses (LDA) for tetanus toxoid (lT)-specificT cells were performed in the presence or absence of exogenousantLCD28 antibody. Addition of antLCD28 yielded no proliferationin the absence of specific antigen. The precursor frequencyfor lT in the CD4⁺ CD45RO⁺ population was –1:4000, whilethe frequency of CD4⁺ CD45RA⁺ T cells specific for lT was 4-to >>20-fold lower. Addition of anti-CD28 antibody didnot significantly alter the apparent precursor frequency forCD45RA⁺ cells but yielded an enhancement of the value for CD45RA⁺cells by 3- to >>5-fold. No enhancement of antigen-specificproliferation by antLCD28 was observed with CD45RA⁺ T cellsderived from cord blood, although phytohemagglutinin responsesof these cells were amplified by CD28 antibody. These resultsindicate that conventional LDA underestimate the true precursorfrequency of antigen-specific cells within the adult CD45RA⁺population and support the possibility that a small number ofcells revert from a primed (CD45RO⁺) to an unprimed (CD45RA⁺)state. The majority of memory T cells, however, appear to residein the CD45RO⁺ population     

Phenotypic characterization of regulatory CD4+CD25+ T cells in rats

Phenotypic characterization of regulatory CD4⁺CD25⁺ T cells     

Phenotypic characterization of regulatory CD4+CD25+ T cells in rats

CD25 has become widely used as a marker for a subset of regulatory CD4(+) T cells present in the thymus and periphery of mice, rats and humans. However, CD25 is also expressed on conventionally activated T cells that are not regulatory and not all peripheral regulatory T cells express CD25. The identification of a stable and unique marker for regulatory T cells would therefore be valuable. This study provides a detailed account of the phenotype of CD4(+)CD25(+) regulatory T cells in rats. In the thymus, CD4(+)CD8(-)CD25(+) cells were found to have a more mature phenotype than the corresponding CD4(+)CD8(-)CD25(-) cells with respect to expression of Thy1 (CD90), CD53 and CD44, suggesting that CD25 expression, and perhaps commitment to regulatory function, might be a late event in thymocyte development. CD4(+)CD25(+) cells in both the thymus and periphery were found to have enriched and heterogeneous expression of activation markers such as OX40 (CD134) and OX48 (an antibody determined in this study to be specific for CD86). CD4(+)CD25(+) T cells were also found to have enriched expression of CD80, at both the mRNA and protein level. However, functional studies in vitro and in vivo showed that neither OX40 or CD86 were useful markers for the further subdivision of regulatory T cells. Our studies indicate that, at present, CD25 remains the most useful marker to enrich for regulatory CD4(+) T cells in rats and no further subdivision of the regulatory component of CD4(+)CD25(-)CD45RC(low) T cells has yet been achieved.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Regulated secretion from CD4+ T cells

The regulated secretion of cellular proteins is central to the correct function of many cell types, including immune cells. Lymphocyte control of the storage, transport and exocytosis of immunomodulatory molecules is a highly specialised task triggered by T cell receptor engagement. The regulated secretory pathway in CD8+ T and NK cells has been the focus of much research, and recent advances have provided insight into the molecular mechanisms governing secretory organelle biogenesis, trafficking and killing. By contrast, regulated secretory pathways in CD4+ T cells have not been studied extensively. Aside from their physiological function in normal T cells, components of CD4+ T cell secretory pathways might be implicated in the assembly of HIV-1. Here, we review findings that shed light on CD4+ T cell secretion in health and disease.     

CD8+ alphabeta T cells can mediate late airway responses and airway eosinophilia in rats

BACKGROUND: The function of CD8+ T-cell subsets in mediating late allergic responses is incompletely understood. OBJECTIVE: We sought to test the hypothesis that CD8+ alphabeta T cells are proinflammatory in the airways in vivo by using a well-characterized animal model and the technique of adoptive transfer. METHODS: Brown Norway rats were administered CD8 + alphabeta T cells (10 6 ) intraperitoneally purified from lymph node cells of either naive or ovalbumin (OVA)-sensitized rats and were challenged with aerosolized OVA 2 days later. Control rats were sensitized to 100 mug of OVA in Al(OH) 3 subcutaneously or sham sensitized to saline and were OVA challenged 2 weeks later. RESULTS: The OVA-sensitized and OVA-challenged group and the recipients of OVA-primed CD8+ alphabeta T cells had significant late airway responses calculated from lung resistance measured for an 8-hour period after challenge compared with the naive CD8 + alphabeta T cell-transferred group and the sham-sensitized control group. The number of eosinophils in bronchoalveolar lavage fluid increased in the OVA-sensitized group and the OVA-primed CD8+ alphabeta T-cell recipients compared with numbers in the naive CD8+ alphabeta T-cell recipients and the sham-sensitized control group. IL-4 and IL-5 cytokine mRNA expression in bronchoalveolar lavage fluid increased in the OVA-sensitized group and the OVA-primed CD8+ alphabeta T-cell recipients compared with that in the sham-sensitized group. CONCLUSION: We conclude that antigen-primed CD8 + alphabeta T cells might have a proinflammatory role in allergen-driven airway responses in the rat.     

CD4+CD25+ T cells as immunoregulatory T cells in vitro

We have further characterized the in vitro phenotype and function of anergic and suppressive CD4(+)25(+) T cells. Following TCR ligation, DO.11.10 CD4(+)25(+) T cells suppress the activation of OT-1 CD8(+)25(-) T cells in an antigen nonspecific manner. Although suppression was seen when using a mixture of APC from both parental strains, it was very much more marked when using F1 APC. APC pretreated with, and then separated from CD4(+)25(+) T cells did not have diminished T cell costimulatory function, suggesting that APC are not the direct targets of CD4(+)25(+) T cell regulation. CTLA-4 blockade failed to abrogate suppression by CD4(+)25(+) T cells in mixing experiments. Although CD4(+)25(+) T cells failed to respond following cross-linking of TCR, they could be induced to proliferate following the addition of exogenous IL-2, allowing the generation of a T cell line from CD4(+)25(+) T cells. After the first in vitro restimulation, CD4(+)25(+) T cells were still anergic and suppressive following TCR engagement. However, after three rounds of restimulation, their anergic and suppressive status was abrogated.     

Expansion of CD4+ CD25+ Foxp3+ T cells by bone marrow-derived dendritic cells

Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system and have a crucial role in T-lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4(+) CD25(+) Foxp3(+) T-cell population after co-culture of lymph node cells from BALB/c mice with syngeneic bone marrow-derived DCs. Our results showed an increase in CD4(+) CD25(+) Foxp3(+) T cells after co-culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4(+) CD25(+) Foxp3(+) T-cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)-beta. The presence of allogeneic apoptotic cells induced interleukin (IL)-2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4(+) CD25(+) Foxp3(+) T-cell expansion during co-culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF-beta and IL-2 in the augmentation of the CD4(+) CD25(+) Foxp3(+) population.     

Ethylenecarbodiimide-coupled allogeneic antigen presenting cells induce human CD4+ regulatory T cells

Adoptive transfer of naturally occurring CD4(+)CD25(+) regulatory T cells can tolerize transplantation alloresponses in animal models. However isolation of these cells in sufficient numbers from humans is cumbersome and prone to contamination with alloreactive CD25(+) T cells. Incubation of ethylenecarbodiimide-coupled antigen presenting cells (APC) with na?ve T cells and antigen has been shown to induce tolerance in various experimental models. We therefore investigated whether ECDI-coupled allogeneic APC were able to induce an expandable human CD4(+) Treg population. CD4(+) and CD4(+) CD25(-) cells cultured for 5 days with ECDI-treated human PBMC exhibited potent suppressive capacity in a mixed lymphocyte reaction. Induction of these ECDI-Tregs was associated with up-regulation of Foxp3 mRNA and protein expression and they maintained high expression of CD62L and CD27 as well as low CD127 expression. ECDI-treated APC displayed reduced expression of the co-stimulatory signaling molecules CD40 and CD80, and failed to stimulate proliferation and cytokine secretion in co-cultured CD4(+) T cells. Restimulation in the presence of rapamycin and hrIL-2 led to expansion of ECDI-Tregs with increasing Foxp3 levels and suppressive activity significantly higher than expanded naturally occurring CD4(+)CD25(+) Tregs. In summary these findings support the hypothesis that ECDI-coupled APC can convert na?ve CD4(+) T cells into functional Tregs with different phenotypic characteristics than naturally occurring CD4(+)CD25(+) Tregs. These inducible Tregs could provide a novel approach that might facilitate the translation of ex vivo generated and expanded Tregs into clinical settings.