Long-term effects of intermittent interleukin 2 therapy in patients with HIV infection: characterization of a novel subset of CD4(+)/CD25(+) T cells

The long-term immunologic effects of intermittent interleukin 2 (IL-2) therapy were evaluated in a cross-sectional study by comparing 3 groups: HIV-seronegative volunteers, HIV-infected (HIV(+)) patients receiving highly active antiretroviral therapy (HAART), and HIV(+) patients receiving HAART and intermittent IL-2. Whole-blood immunophenotyping was performed to study expression of the IL-2 receptor chains on T lymphocytes and natural killer cells and to further characterize CD4(+)/CD25(+) T cells. Increased CD25 expression, especially in CD4(+) T cells but also in CD8(+) T cells, without increases in expression of the beta and gamma chains of the IL-2 receptor was detected in the IL-2 group. Up to 79% of naive CD4(+) T cells (median, 61%) from patients in the IL-2 group expressed CD25, and the number of naive CD4(+)/CD25(+) T cells correlated positively with both the total and naive CD4(+) T-cell counts. A discrete population of CD45 double intermediate RA(+)/RO(+) CD4(+) cells was also preferentially expanded in the IL-2 group, and the number of these cells strongly correlated with the total CD4(+) count. Despite increases in CD25 expression, T lymphocytes from patients treated with IL-2 did not have increased expression of early (CD69) or late (CD95) activation markers or evidence of recent proliferation (Ki67). Both CD4(+)/CD25(+) and CD4(+)/CD25(-) cells from IL-2-treated HIV(+) patients proliferated in response to mitogens, specific antigens, and T-cell-receptor-mediated stimuli. Thus, intermittent administration of IL-2 in HIV(+) patients leads to preferential expansion of a unique subset of CD4(+) T cells that may represent a critical population in T-cell homeostasis.     

Activation of naturally occurring lung CD4(+)CD25(+) regulatory T cells requires CD8 and MHC I interaction

Naturally occurring Foxp3(+)CD4(+)CD25(+) T cells (nTregs) isolated from lungs of naive mice regulate allergic airway hyperresponsiveness (AHR) and inflammation. Here, we demonstrate the critical requirement for engagement of MHC class I on CD4(+)CD25(+) T cells by CD8 for the functional activation of these nTregs. Suppression of allergen-induced AHR and inflammation by nTregs was abolished in mice treated with anti-CD8. Correspondingly, decreased levels of IL-10 and TGF-beta and increased levels of Th2 cytokines in bronchoalveolar lavage were detected in these treated mice. Similarly, nTregs isolated from beta2m(-/-) mice or from mice treated with anti-MHC I antibody in vitro before intratracheal transfer failed to modulate AHR or inflammation. Coculture of nTregs with CD8(+) T cells increased IL-10 and TGF-beta. Addition of anti-MHC I or anti-CD8 reduced IL-10 and TGF-beta. These results demonstrate that functional activation of nTregs requires the interaction between MHC I on CD4(+)CD25(+) T cells and CD8.     

CD4+ CD25+调节性T细胞在哮喘患者体内的功能变化及其意义

目的探讨CD_4~ CD_(25)~ 调节性T细胞在哮喘患者体内的功能变化。进一步探讨哮喘的发病机制。方法使用免疫磁珠分选正常对照组和哮喘患者的CD_4~ CD_(25)~ 调节性T细胞;流式细胞仪分析其纯度;分别用变应原或联合应用抗CD3单抗和抗CD28单抗,刺激两种来源的CD_4~ CD_(25)~ 调节性T细胞和CD_4~ CD_(25)~-5T细胞,ELISA、3H-TdR等检测CD_4~ CD_(25)~ 调节性T细胞功能。结果:来源于哮喘患者的CD4 CD2 5调节性T细胞不能抑制变应原对CD_4~ CD_(25)~-T细胞的活化增殖,而能抑制抗CD3和抗CD28单抗对CD_4~ CD_(25)~-T细胞的刺激作用。结论哮喘患者体内CD_4~ CD_(25)~ 调节性T细胞存在功能缺陷。     

CD4+CD25+ regulatory T cells control CD8+ T-cell effector differentiation by modulating IL-2 homeostasis

CD4(+)CD25(+) regulatory T cells (Treg) play a crucial role in the regulation of immune responses. Although many mechanisms of Treg suppression in vitro have been described, the mechanisms by which Treg modulate CD8(+) T cell differentiation and effector function in vivo are more poorly defined. It has been proposed, in many instances, that modulation of cytokine homeostasis could be an important mechanism by which Treg regulate adaptive immunity; however, direct experimental evidence is sparse. Here we demonstrate that CD4(+)CD25(+) Treg, by critically regulating IL-2 homeostasis, modulate CD8(+) T-cell effector differentiation. Expansion and effector differentiation of CD8(+) T cells is promoted by autocrine IL-2 but, by competing for IL-2, Treg limit CD8(+) effector differentiation. Furthermore, a regulatory loop exists between Treg and CD8(+) effector T cells, where IL-2 produced during CD8(+) T-cell effector differentiation promotes Treg expansion.     

CD70+ non-Hodgkin lymphoma B cells induce Foxp3 expression and regulatory function in intratumoral CD4+CD25 T cells

Foxp3 expression was initially thought to be restricted to the CD4(+)CD25(+) regulatory T-cell population. However, recent studies suggest that forkhead box P3 (Foxp3) is expressed in CD4(+)CD25(-) T cells in aged mice. In the present study in B-cell non-Hodgkin lymphoma (NHL), we found that a subset of intratumoral but not peripheral blood CD4(+)CD25(-) T cells, comprising about 15% of intratumoral CD4(+) T cells, express Foxp3 and are capable of suppressing the proliferation of autologous infiltrating CD8(+) T cells. In vitro activation with OKT3/anti-CD28 antibody (Ab) or dendritic cells (DCs) induced Foxp3 expression in a subset of these CD4(+)CD25(-)Foxp3(-) T cells. We found that the presence of lymphoma B cells during activation augmented activation-induced Foxp3 expression in CD4(+)CD25(-) T cells. We also found that CD70(+) lymphoma B cells significantly contributed to the activation-induced Foxp3 expression in intratumoral CD4(+)CD25(-) T cells. Furthermore, the blockade of CD27-CD70 interaction by anti-CD70 Ab abrogated lymphoma B-cell-mediated induction of Foxp3 expression in intratumoral CD4(+)CD25(-) T cells. Taken together, these studies reveal a novel role for NHL B cells in the development of intratumoral regulatory T cells.     

Blockade of CD86 and CD40 induces alloantigen-specific immunoregulatory T cells that remain anergic even after reversal of hyporesponsiveness

The generation of immunoregulatory T cells that block the B7(CD86/CD80)-CD28 and/or CD40-CD154 costimulatory pathways has great potential for the induction of long-term transplantation tolerance. In a human polyclonal in vitro model, combined monoclonal antibody (mAb) blocking of the costimulatory ligands CD40 and CD86 lead to allospecific T-cell anergy that cannot be reversed by antigenic rechallenge in the presence of IL-2. Although antigenic restimulation with IL-2 restored the proliferative response, subsequent antigenic restimulation of the restored anergic cells in a tertiary mixed lymphocyte culture still resulted in nonresponsiveness. Importantly, these anergic T cells suppress the response of naive alloreactive T cells in an antigen-specific way via linked recognition. Suppression may partially depend on local IL-10 production, while transforming growth factor-beta (TGF-beta) did not play a role. Irrespective of the monoclonal antibody combination used, blast formation occurred in a subset of CD4(+) cells. These cells were characterized by a sustained CD45RA expression, an increased T-cell receptor density, and a lower level of CD4 expression. A reduced number of CD45RO(+)/CD8(+) T cells was observed whenever anti-CD86 was combined with anti-CD40, which was reflected by an even more attenuated cytotoxic T-cell function. This indicates the importance of CD40-CD154 in the generation of cytotoxic T cells in this transplantation model. We hypothesize that in our model, anergy is induced in the CD4(+) T-cell subset, whereby CD8(+) cytotoxic effector function is impaired by the lack of both CD40-CD154 signaling and cytokine-mediated help. This costimulatory ligand-directed mAb approach might well be used for the ex vivo generation of antigen-specific immunoregulatory T cells applicable in adoptive immunotherapy.     

Adenovirus type-35 vectors block human CD4+ T-cell activation via CD46 ligation

Recombinant adenoviruses (rAds) based on types 5 (rAd5) and 35 (rAd35) have emerged as important vaccine delivery vectors in clinical testing for a variety of pathogens. A major difference between these vectors is their binding to cellular receptors used for infection. Whereas rAd5 binds coxsackie-adenovirus receptor (CAR), rAd35 binds the complement regulatory protein CD46. Although rAd35 infected and phenotypically matured human blood dendritic cells (DCs) more efficiently than rAd5, we show here that rAd35 markedly suppressed DC-induced activation of naive CD4(+) T cells. rAd35 specifically blocked both DCs and anti-CD3/CD28 mAb-induced naive T-cell proliferation and IL-2 production. This effect was also observed in CD4(+) memory T cells but to a lesser extent. The suppression occurred by rAd35 binding to CD46 on T cells and was independent of infection. CD46 engagement with mAb mimicked the effects of rAd35 and also led to deficient NF-κB nuclear translocation. In contrast, rAd5 and rAd35 vectors with ablated CD46 binding did not inhibit T-cell activation. Our findings provide insights into the basic biology of adenoviruses and indicate that CD46 binding may have an impact on the generation of primary CD4(+) T-cell responses by Ad35.     

Dysregulation of CD4+CD25+CD127lowFOXP3+ regulatory T cells in HIV-infected pregnant women

Pregnancy represents a major challenge to immunologic tolerance. How the fetal "semiallograft" evades maternal immune attack is unknown. Pregnancy success may involve alteration of both central (thymic) and peripheral tolerance mechanisms. HIV infection is characterized by CD4(+) T-cell depletion, chronic immune activation, and altered lymphocyte subsets. We studied immunologic consequences of pregnancy in 20 HIV-infected women receiving highly active antiretroviral therapy (HAART), and for comparison in 16 HIV-negative women. Lymphocyte subsets, thymic output, and cytokine profiles were measured prospectively during pregnancy and postpartum. A significant expansion of CD4(+)CD25(+)CD127(low)FoxP3(+) regulatory T cells indicating alteration of peripheral tolerance was seen during second trimester, but only in HIV-negative women. HIV-infected women had lower CD4 counts, lower thymic output and Th-2 cytokines, and more immune activation at all time points compared with controls. Immune activation was decreased in HIV-infected patients during pregnancy. In contrast, CD4 counts were increased in both groups. In conclusion, the study does not indicate that pregnancy adversely affects the immunologic course of HIV infection. However, despite HAART during pregnancy, HIV-infected women display different immunologic profiles from HIV-negative women, which may have importance for the induction of fetal-maternal tolerance and in part explain the increased risk of abortion in HIV-infected women.     

Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects

We have genetically engineered CD4(+) and CD8(+) T cells with human immunodeficiency virus (HIV) specificity by inserting a gene, CD4zeta, containing the extracellular domain of human CD4 (which binds HIV env) linked to the zeta (zeta) chain of the T-cell receptor (which mediates T-cell activation). Twenty-four HIV-positive subjects received a single infusion of 2 to 3 x 10(10) autologous CD4zeta-modified CD4(+) and CD8(+) T cells administered with (n = 11) or without (n = 13) interleukin-2 (IL-2). Subjects had CD4 counts greater than 50/microL and viral loads of at least 1000 copies/mL at entry. T cells were costimulated ex vivo through CD3 and CD28 and expanded for approximately 2 weeks. CD4zeta was detected in 1% to 3% of blood mononuclear cells at 8 weeks and 0.1% at 1 year after infusion, and survival was not enhanced by IL-2. Trafficking of gene-modified T cells to bulk rectal tissue and/or isolated lamina propria lymphocytes was documented in a subset of 5 of 5 patients at 14 days and 2 of 3 at 1 year. A greater than 0.5 log mean decrease in rectal tissue-associated HIV RNA was observed for at least 14 days, suggesting compartmental antiviral activity of CD4zeta T cells. CD4(+) counts increased by 73/microL at 8 weeks in the group receiving IL-2. There was no significant mean change in plasma HIV RNA or blood proviral DNA in either treatment arm. This sustained, high-level persistence of gene-modified T cells demonstrates the feasibility of ex vivo T-cell gene therapy in HIV-infected adults and suggests the importance of providing HIV-specific T-helper function.     

An anti-CD45RO immunotoxin kills latently infected human immunodeficiency virus (HIV) CD4 T cells in the blood of HIV-positive persons.

Highly active antiretroviral therapy has decreased the morbidity and mortality of human immunodeficiency virus (HIV) infection, but latently infected cells remain for prolonged periods. CD4(+) CD45RO(+) T cells are a major latent virus reservoir in HIV-infected persons. Replication-competent, latently HIV-infected T cells can be generated in vitro by infecting peripheral blood mononuclear cells with HIV and then eliminating the HIV-producing cells with an anti-CD25 immunotoxin (IT). The CD25(-) latently infected cells then can be eliminated with an anti-CD45RO IT. This study determined whether this IT also could kill latently infected CD4 T cells from HIV-infected persons with or without detectable plasma viremia. The results show that ex vivo treatment of cells from HIV-positive persons by anti-CD45RO IT reduces the frequency of both productively and latently infected cells. In contrast, CD4(+) CD45RA(+) naive T cells and a proportion of CD4(+) CD45RO(lo) memory T cells are spared.     

Role for granulocyte colony-stimulating factor in the generation of human T regulatory type 1 cells

Granulocyte colony-stimulating factor (G-CSF) may affect T-cell homeostasis by multiple mechanisms, inducing polarization of cytokine secretion, inhibition of T-cell proliferation, and enhancement of T-cell apoptosis. We analyzed the production of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1) by T cells from healthy volunteer donors treated with recombinant human G-CSF. Highly purified CD4(+) T cells obtained before and after G-CSF administration (pre-G and post-G, respectively) were activated using the allogeneic mixed leukocyte reaction. Post-G CD4(+) T cells produced high levels of IL-10 but undetectable levels of IL-2 and IL-4, whereas the level of TGF-beta1 release was comparable to that of pre-G CD4(+) T cells. Notably, post-G CD4(+) T cells proliferated poorly in response to alloantigens and to recall antigens and suppressed the proliferation of autologous CD4(+) T cells in a cell contact-independent and an antigen-nonspecific manner. TGF-beta1 and IL-10 were not dispensable for post-G CD4(+) T cells to mediate suppression, as shown by neutralization studies. Compared with pre-G CD4(+) T cells, alloantigen-activated post-G CD4(+) T cells preferentially expressed markers associated with memory T cells, in conjunction with reduced levels of CD28 and CD62L. Collectively, these data demonstrate that CD4(+) T cells exposed to G-CSF in vivo acquire the properties of T regulatory (Tr) cells once triggered in vitro through the T-cell receptor, including a peculiar cytokine production profile (IL-10(++)TGF-beta1(+)IL-2(low/-)IL-4(low/-)), an intrinsic low proliferative capacity, and a contact-independent suppression of antigen-driven proliferation. Tr cells generated ex vivo after exposure to G-CSF might be clinically relevant for transplantation medicine and for the treatment of human immune-mediated diseases.     

CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.     

Proinflammatory mediator-induced reversal of CD4+,CD25+ regulatory T cell-mediated suppression in rheumatoid arthritis

OBJECTIVE: We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg-mediated suppression. METHODS: Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme-linked immunosorbent assay. Magnetically sorted CD4+,CD25- and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti-CD3 monoclonal antibody (mAb) and autologous antigen-presenting cells, in the absence or presence of anti-CD28 mAb or the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), or IL-7. RESULTS: Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB-derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti-CD28 mAb to cocultures of CD4+,CD25- and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg-mediated suppression in both PB and SF. Furthermore, IL-7 and, to a limited extent, TNFalpha, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg-mediated suppression. In contrast, IL-6 did not influence Treg-mediated suppression. CONCLUSION: Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.     

Perturbations in B cell responsiveness to CD4+ T cell help in HIV-infected individuals

HIV infection induces a wide array of B cell dysfunctions. We have characterized the effect of plasma viremia on the responsiveness of B cells to CD4(+) T cell help in HIV-infected patients. In HIV-negative donors, B cell proliferation correlated with CD154 expression on activated CD4(+) T cells and with the availability of IL-2, whereas in HIV-infected viremic patients, reduced B cell proliferation was observed despite normal CD154 expression on activated CD4(+) T cells. Reduced triggering of B cells by activated CD4(+) T cells was clearly observed in HIV-infected viremic patients compared with aviremic patients with comparable CD4(+) T cell counts, and a dramatic improvement in B cell function was observed in patients whose plasma viremia was controlled by effective antiretroviral therapy. The degree of B cell dysfunction in viremic patients correlated strongly with the inability of B cells to express CD25 in response to activated CD4(+) T cells, resulting in an inability to mount a normal proliferative response to IL-2. Similar defects in responsiveness to IL-2 were observed in the B cells of HIV-infected viremic patients in the context of B cell receptor stimulation. These data provide new insight into the mechanisms associated with ineffective humoral responses in HIV disease.     

Suppression of autoreactive T-cell response to glycoprotein IIb/IIIa by blockade of CD40/CD154 interaction: implications for treatment of immune thrombocytopenic purpura

The potential immunosuppressive effect of an anti-CD154 monoclonal antibody (mAb) on the pathogenic autoreactive T-cell response was evaluated using an in vitro culture system with glycoprotein IIb/IIIa (GPIIb/IIIa)-reactive T cells from patients with immune thrombocytopenic purpura (ITP). The anti-CD154 mAb did not inhibit T-cell proliferation, but suppressed anti-GPIIb/IIIa antibody production, in bulk peripheral blood mononuclear cell cultures stimulated with GPIIb/IIIa. Repeated antigenic stimulation of GPIIb/IIIa-reactive CD4(+) T-cell lines in the presence of anti-CD154 mAb resulted in the loss of proliferative capacity and helper function for promoting anti-GPIIb/IIIa antibody production. These anergic T-cell lines showed a cytokine profile of low interferon gamma and high interleukin 10 and suppressed anti-GPIIb/IIIa antibody production. Our results indicate that blockade of the CD40/CD154 interaction induces generation of autoantigen-specific anergic CD4(+) T cells with regulatory function and could be a therapeutic option for suppressing pathogenic autoimmune responses in patients with ITP.     

CD25+ regulatory T cells isolated from HIV-infected individuals suppress the cytolytic and nonlytic antiviral activity of HIV-specific CD8+ T cells in vitro

HIV infection is characterized by CD4(+) T cell depletion and progressive immune dysfunction; particularly impacted are HIV-specific T cell responses. An important component of immune-mediated control of HIV replication, killing of infected cells, appears to be impaired, in part due to poor cytolytic activity of HIV-specific cytotoxic T cells (CTL). In vitro, several functions of HIV-specific T cells, such as cytokine production, can be enhanced by the depletion of the immunosuppressive CD25(+) FoxP3(+) CD4(+) regulatory (Treg) cell subset. However, the effect of CD25(+) Treg cells on virus-specific cytolytic activity in the context of HIV or any human viral infection has not been investigated. The present study demonstrates that CD25(+) Treg cells isolated from the peripheral blood of HIV-infected subjects significantly suppress HIV Gag-specific cytolytic activity in vitro. In addition, CD25(+) Treg cells suppress effector function (coexpression of TNF-alpha and IFN-gamma) of HIV-specific CD8(+) T cells that proliferate in response to HIV antigen. Finally, the secretion of HIV-inhibitory CC-chemokines by HIV-specific and nonspecific CD8(+) T cells is significantly reduced in the presence of CD25(+) Treg cells. These data suggest that CD25(+) Treg-mediated suppression of the antiviral activity of HIV-specific CD8(+) T cells could impact the ability of HIV-infected individuals to control HIV replication in vivo.