Function Of Human CD4 Research Articles

Gut microbial metabolites facilitate anticancer therapy efficacy by modulating cytotoxic CD8+ T cell immunity

Recent studies in both mice and humans have suggested that gut microbiota could modulate tumor responsiveness to chemo- or immunotherapies. However, the underlying mechanism is not clear yet. Here, we found that gut microbial metabolites, especially butyrate, could promote the efficacy of oxaliplatin by modulating CD8+ Tcell function in the tumor microenvironment. Butyrate treatment directly boosted the antitumor cytotoxic CD8+ Tcell responses both invitro and invivo in an ID2-dependent manner by promoting the IL-12 signaling pathway. In humans, the oxaliplatin responder cancer patients exhibited a higher amount of serum butyrate than did non-responders, which could also increase ID2 expression and function of human CD8+ Tcells. Together, our findings suggest that the gut microbial metabolite butyrate could promote antitumor therapeutic efficacy through the ID2-dependent regulation of CD8+ Tcell immunity, indicating that gut microbial metabolites could be effective as a part of cancer therapy.

Ionizing radiation modulates the phenotype and function of human CD4+ induced regulatory T cells

BackgroundThe use of immunotherapy strategies for the treatment of advanced cancer is rapidly increasing. Most immunotherapies rely on induction of CD8+ tumor-specific cytotoxic T cells that are capable of directly killing cancer cells. Tumors, however, utilize a variety of mechanisms that can suppress anti-tumor immunity. CD4+ regulatory T cells can directly inhibit cytotoxic T cell activity and these cells can be recruited, or induced, by cancer cells allowing escape from immune attack. The use of ionizing radiation as a treatment for cancer has been shown to enhance anti-tumor immunity by several mechanisms including immunogenic tumor cell death and phenotypic modulation of tumor cells. Less is known about the impact of radiation directly on suppressive regulatory T cells. In this study we investigate the direct effect of radiation on human TREG viability, phenotype, and suppressive activity.ResultsBoth natural and TGF-β1-induced CD4+ TREG cells exhibited increased resistance to radiation (10 Gy) as compared to CD4+ conventional T cells. Treatment, however, decreased Foxp3 expression in natural and induced TREG cells and the reduction was more robust in induced TREGS. Radiation also modulated the expression of signature iTREG molecules, inducing increased expression of LAG-3 and decreased expression of CD25 and CTLA-4. Despite the disconcordant modulation of suppressive molecules, irradiated iTREGS exhibited a reduced capacity to suppress the proliferation of CD8+ T cells.ConclusionsOur findings demonstrate that while human TREG cells are more resistant to radiation-induced death, treatment causes downregulation of Foxp3 expression, as well as modulation in the expression of TREG signature molecules associated with suppressive activity. Functionally, irradiated TGF-β1-induced TREGS were less effective at inhibiting CD8+ T cell proliferation. These data suggest that doses of radiotherapy in the hypofractionated range could be utilized to effectively target and reduce TREG activity, particularly when used in combination with cancer immunotherapies.

Modulation of Phenotype and Function of Human CD4+CD25+ T Regulatory Lymphocytes Mediated by cAMP-Elevating Agents.

We have shown that cholera toxin (CT) and other cyclic AMP (cAMP)-elevating agents induce upregulation of the inhibitory molecule CTLA-4 in human resting CD4+ T lymphocytes, which following the treatment acquired suppressive functions. In this study, we evaluated the effect of cAMP-elevating agents on human CD4+CD25+ T cells, which include the T regulatory cells (Tregs) that play a pivotal role in the maintenance of immunological tolerance. We found that cAMP-elevating agents induce upregulation of CTLA-4 in CD4+CD25− and further enhance its expression in CD4+CD25+ T cells. We observed an increase of two isoforms of mRNA coding for the membrane and the soluble CTLA-4 molecules, suggesting that the regulation of CTLA-4 expression by cAMP is at the transcriptional level. In addition, we found that the increase of cAMP in CD4+CD25+ T cells converts the CD4+CD25+Foxp3− T cells in CD4+CD25+Foxp3+ T cells, whereas the increase of cAMP in CD4+CD25− T cells did not upregulate Foxp3 in the absence of activation stimuli. To investigate the function of these cells, we performed an in vitro suppression assay by culturing CD4+CD25+ T cells untreated or pre-treated with CT with anti-CD3 mAbs-stimulated autologous peripheral blood mononuclear cell. We found that CT enhances the inhibitory function of CD4+CD25+ T cells, CD4+, and CD8+ T cell proliferation and IFNγ production are strongly inhibited by CD4+CD25+ T cells pre-treated with cAMP-elevating agents. Furthermore, we found that CD4+CD25+ T lymphocytes pre-treated with cAMP-elevating agents induce the upregulation of CD80 and CD86 co-stimulatory molecules on immature dendritic cells (DCs) in the absence of antigenic stimulation, however without leading to full DC maturation. These data show that the increase of intracellular cAMP modulates the phenotype and function of human CD4+CD25+ T cells.

Differential effect of HLA class-I versus class-II transgenes on human T and B cell reconstitution and function in NRG mice.

Humanized mice expressing Human Leukocyte Antigen (HLA) class I or II transgenes have been generated, but the role of class I vs class II on human T and B cell reconstitution and function has not been investigated in detail. Herein we show that NRG (NOD.RagKO.IL2RγcKO) mice expressing HLA-DR4 molecules (DRAG mice) and those co-expressing HLA-DR4 and HLA-A2 molecules (DRAGA mice) did not differ in their ability to develop human T and B cells, to reconstitute cytokine-secreting CD4 T and CD8 T cells, or to undergo immunoglobulin class switching. In contrast, NRG mice expressing only HLA-A2 molecules (A2 mice) reconstituted lower numbers of CD4 T cells but similar numbers of CD8 T cells. The T cells from A2 mice were deficient at secreting cytokines, and their B cells could not undergo immunoglobulin class switching. The inability of A2 mice to undergo immunoglobulin class switching is due to deficient CD4 helper T cell function. Upon immunization, the frequency and cytotoxicity of antigen-specific CD8 T cells in DRAGA mice was significantly higher than in A2 mice. The results indicated a multifactorial effect of the HLA-DR4 transgene on development and function of human CD4 T cells, antigen-specific human CD8 T cells, and immunoglobulin class switching.

ICOS Regulates the Generation and Function of Human CD4+ Treg in a CTLA-4 Dependent Manner

Inducible co-stimulator (ICOS) is a member of CD28/Cytotoxic T-lymphocyte Antigen-4 (CTLA-4) family and broadly expressed in activated CD4+ T cells and induced regulatory CD4+ T cells (CD4+ iTreg). ICOS-related signal pathway could be activated by the interaction between ICOS and its ligand (ICOSL). In our previous work, we established a cost-effective system to generate a novel human allo-antigen specific CD4hi Treg by co-culturing their naïve precursors with allogeneic CD40-activated B cells in vitro. Here we investigate the role of ICOS in the generation and function of CD4hi Treg by interrupting ICOS-ICOSL interaction with ICOS-Ig. It is found that blockade of ICOS-ICOSL interaction impairs the induction and expansion of CD4hi Treg induced by allogeneic CD40-activated B cells. More importantly, CD4hi Treg induced with the addition of ICOS-Ig exhibits decreased suppressive capacity on alloantigen-specific responses. Dysfunction of CD4hi Treg induced with ICOS-Ig is accompanied with its decreased exocytosis and surface CTLA-4 expression. Through inhibiting endocytosis with E64 and pepstatin A, surface CTLA-4 expression and suppressive functions of induced CD4hi Treg could be partly reversed. Conclusively, our results demonstrate the beneficial role of ICOS-ICOSL signal pathway in the generation and function of CD4hi Treg and uncover a novel relationship between ICOS and CTLA-4.

Differential role of all-transretinoic acid in promoting the development of CD4+ and CD8+ regulatory T cells

It is known that ATRA promotes the development of TGF-β-induced CD4(+)Foxp3(+) iTregs, which play a vital role in the prevention of autoimmune diseases; however, the role of ATRA in facilitating the differentiation and function of CD8(+)Foxp3(+) iTregs remains elusive. Using a head-to-head comparison, we found that ATRA promoted expression of Foxp3 and development of CD4(+) iTregs, but it did not promote Foxp3 expression on CD8(+) cells. Using a standard in vitro assay, we demonstrated that CD8(+) iTregs induced by TGF-β and ATRA were not superior to CD8(+) iTregs induced by TGF-β alone. In cGVHD, in a typical lupus syndrome model where DBA2 spleen cells were transferred to DBA2xC57BL/6 F1 mice, we observed that both CD8(+) iTregs induced by TGF-β and ATRA and those induced by TGF-β alone had similar therapeutic effects. ATRA did not boost but, conversely, impaired the differentiation and function of human CD8(+) iTregs. CD8(+) cells expressed the ATRA receptor RAR and responded to ATRA, similar to CD4(+) cells. We have identified the differential role of ATRA in promoting Foxp3(+) Tregs in CD4(+) and CD8(+) cell populations. These results will help to determine a protocol for developing different Treg cell populations and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation.

Efficient peripheral construction of functional human regulatory CD4+CD25highFoxp3+ T cells in NOD/SCID mice grafted with fetal human thymus/liver tissues and CD34+ cells

Regulatory T cells, especially CD4+CD25+ regulatory T cells are critical regulators of immune tolerance in humans and mice. Mice with humanized immunity have been developed by various transplantation strategies of human tissues or cells related to immunity, which are being extensively applied in biomedical research. However, it is unclear whether human CD4+CD25+ regulatory T cells can normally develop in human thymic grafts and efficiently populate in the periphery in NOD/SCID mouse recipients. In human thymic grafts, high percentage of mature human CD4+CD25high regulatory T cells was detected. Human CD4+CD25+ regulatory T cells maturing in fetal human thymus grafts could subsequently output to the periphery of NOD/SCID mouse recipients. Importantly, these cells exhibited Foxp3+CD45RO+CTLA4+CD127− phenotype, similarly to those in healthy individuals. In addition, human CD4+CD25+ regulatory T cells maturing in human thymic grafts suppressed proliferative response of CD4+CD25− T cells to allogeneic antigens, though the peripheral CD4+CD25+ regulatory T cells in fetal human thymus-grafted NOD/SCID mice showed somewhat decreased immunosuppressive ability compared with normal CD4+CD25+ regulatory T cells. Thus, this humanized animal model is suitable for examining development and function of human CD4+CD25+ regulatory T cells in vivo.

A Dual Action of Rheumatoid Arthritis Synovial Fibroblast IL-15 Expression on the Equilibrium between CD4+CD25+ Regulatory T Cells and CD4+CD25− Responder T Cells

We previously described that fibroblast-like cells from the synovium of rheumatoid arthritis patients (RASFib) constitutively express intracellular and surface IL-15, which induces activation of cocultured T cells. Our objective was to study the effect of RASFib IL-15 expression on the function of human CD4(+)CD25(+) regulatory T cells (Treg). RASFib, through their constitutive IL-15 expression, were able to induce the proliferation of human Tregs stimulated through their TCR, and at the same time potentiated their suppressive action on the cytokine secretion of CD4(+)CD25(-) responder T cells (Tresp). In parallel, constitutive RASFib IL-15 expression mediated an up-regulated response of Tresp. Subsequently, total CD4(+) T cells, containing natural proportions of Treg and Tresp, secreted an increased amount of pathogenic cytokines when cocultured with RASFib despite the presence of proliferating Treg with superior regulatory potency. In summary, RASFib IL-15 exerts a dual action on the equilibrium between Treg and Tresp by potentiating the suppressive effect of Treg while augmenting the proinflammatory action of Tresp; the result is a shift of the Treg/Tresp balance toward a proinflammatory state. This alteration of the Treg/Tresp equilibrium is not observed in the presence of osteoarthritis synovial fibroblasts or dermal fibroblasts, which do not constitutively express surface IL-15. Additionally, Treg with superior suppressive potency were present in the peripheral blood and the synovial fluid of RA patients, but this enhanced immunoregulatory activity was not able to overcome the increased secretion of pathogenic cytokines by RA-Tresp, indicating that rheumatoid arthritis patients demonstrate an altered Treg/Tresp equilibrium in vivo.

The role of retinoic acid‐related orphan receptor variant 2 and IL‐17 in the development and function of human CD4+ T cells

Th17 cells are defined by their capacity to produce IL-17, and are important mediators of inflammation and autoimmunity. Human Th17 cells express high levels of the retinoic acid-related orphan receptor variant 2 (RORC2), but it is currently unclear whether expression of this transcription factor alone is sufficient to recapitulate all the known properties of Th17 cells. We used lentivirus-mediated transduction to investigate the role of RORC2 in defining aspects of the human Th17 cell lineage. Expression of RORC2 induced production of IL-17A, IL-22, IL-6 and TNF-alpha, a Th17-cell-associated chemokine receptor profile and upregulation of CD161. RORC2-transduced T cells were hypo-responsive to TCR-mediated stimulation, a property shared with ex vivo Th17 cells and overcome by addition of exogenous IL-2 or IL-15. Co-culture experiments revealed that RORC2-expressing cells were partially resistant to Treg cells since production of IL-17 and proliferation were not suppressed. Evidence that IL-17 stimulates CD4(+) T cells to produce IL-2 and proliferate suggested that the resistance of Th17 cells to Treg-mediated suppression may be partly attributed to IL-17 itself. These findings demonstrate that expression of RORC2 in T cells has functional consequences beyond altering cytokine production and provides insight into the factors regulating the development of human Th17 cells.

Changes of phenotype and function of human CD4+ CD25− T cells induced by transfection of Foxp3

The aim of this paper is to explore the effects of transfection of Foxp3 gene on the phenotype and function of naive CD4+ T cells. The pMSCV-Foxp3 retroviral vector encoding Foxp3 gene was transduced into the PT67 packaging cell line. Virus-containing supernatant was applied to differentiate CD4+CD25− T cells. The resulting cells were sorted with flow cytometry. The expressions of CD25, CD127, CTLA-4 and the proliferation of transfected T cells were examined. The effect of transfected CD4+ T cells on the proliferation and cytokine production of CD4+CD25− T cells was examined. Foxp3-gene transfected CD4+ T cells could express Foxp3 and transfection of Foxp3 gene up-regulated the expressions of CD25 and CTLA-4, but down-regulated CD127 expression. After transfection, the proliferation of CD4+ T cells was eliminated. Transfected T cells inhibited the proliferation of CD4+CD25− T cells. CD4+CD25− T cells acquired a regulatory phenotype and function after it was transduced with the Foxp3 gene. This suggested a key role of Foxp3 in the generation of CD4+CD25+ regulatory T cells.

Reduced CD4+ T-cell-specific gene expression in human type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) in humans is characterized by the T-cell-dependent destruction of the insulin producing pancreatic beta cells; however, the precise pathogenesis of the disease, especially the initiation of pathologic immune response, is still largely unknown. We hypothesized that the function of human CD4+ T cells is altered in T1DM and analyzed unstimulated human peripheral blood CD4+ T-cell gene expression. We used a novel three-way comparison of DNA microarray data of CD4+ T cells isolated from patients with new onset T1DM, patients with long-term Type 2 diabetes (T2DM), and from healthy control subjects in order to eliminate any possible influence of glucose homeostasis on our findings. We analyzed the T1DM specific gene-expression changes and their functional relevance to T1DM autoimmunity. Our genetic and functional data show that T1DM CD4+ T cells are down-regulated specifically affecting key immune functions and cell cycle. Histone deacetylase gene expression, a key regulator of epigenetic modification is also reduced. The CD4+ T cells showed impaired function, including an abnormal immune response, which may be a key element that leads to the breakdown of self-tolerance.

Controlling the Generation and Function of Human CD8+ Memory T Cells In Vitro with Immunosuppressants

Memory T cells play a pivotal role in acute and chronic rejection of transplanted organs. Novel therapies such as alemtuzumab and the identification of heterologous memory have highlighted their importance following transplantation. Unlike naive T cells, the influence of immunosuppressive agents on the de novo generation of memory T cells and on the function of pre-existing memory T cells is poorly understood. CD8 effector memory T cells (Tem, CD45RA, CCR7) were generated by stimulating peripheral blood mononuclear cells for five days with anti-CD3 and anti-CD28 monoclonal antibodies, followed by nine days of rest. The influence of immunosuppression on this was assessed by flow cytometry. Tem that had been naturally formed in vivo were used to study the influence of the same agents on the function (intracellular interferon-gamma production) of Tem cells. Whereas all the immunosuppressive agents tested inhibited the expansion of CD8 Tem population by influencing their proliferation and apoptosis, the calcineurin inhibitors were better at controlling their function. Furthermore, Tem generated despite the presence of tacrolimus were functional and did not exhibit drug resistance. Immunosuppressive agents differ in their ability to control the generation and function of CD8 Tem; the calcineurin inhibitors being the most efficacious. This may be of importance when tailoring immunosuppressants for transplant recipients, particularly in those where there is pre-existing immunological memory or in those receiving T-cell depleting therapies, which skews the immune repertoire towards memory.

IL-2 administration increases CD4+CD25hi Foxp3+ regulatory T cells in cancer patients

AbstractInterleukin-2 (IL-2) is historically known as a T-cell growth factor. Accumulating evidence from knockout mice suggests that IL-2 is crucial for the homeostasis and function of CD4+CD25+ regulatory T cells in vivo. However, the impact of administered IL-2 in an immune intact host has not been studied in rodents or humans. Here, we studied the impact of IL-2 administration on the frequency and function of human CD4+CD25hi T cells in immune intact patients with melanoma or renal cancer. We found that the frequency of CD4+CD25hi T cells was significantly increased after IL-2 treatment, and these cells expressed phenotypic markers associated with regulatory T cells. In addition, both transcript and protein levels of Foxp3, a transcription factor exclusively expressed on regulatory T cells, were consistently increased in CD4 T cells following IL-2 treatment. Functional analysis of the increased number of CD4+CD25hi T cells revealed that this population exhibited potent suppressive activity in vitro. Collectively, our results demonstrate that administration of high-dose IL-2 increased the frequency of circulating CD4+CD25hi Foxp3+ regulatory T cells. Our findings suggest that selective inhibition of IL-2-mediated enhancement of regulatory T cells may improve the therapeutic effectiveness of IL-2 administration. (Blood. 2006;107:2409-2414)

Human CD8+ CTL recognition and in vitro lysis of herpes simplex virus-infected cells by a non-MHC restricted mechanism.

Cytotoxic T lymphocytes (CTL) are important for the recognition and lysis of virally infected cells, but their effectiveness can be limited by viral immune evasion mechanisms. We investigated the immunophenotype and function of human CD8+ T cells raised in response to herpes simplex virus (HSV). The expanded population contained cells of an activated and mature phenotype, as determined by the expressions of CD25, CD45RO, CD57, CD95 and HLA-DR. Cultured cells also expressed CD45RA. These cells lysed autologous and allogeneic HSV-infected lymphoblastoid cell line (LCL) targets via a non-major histocompatibility complex (MHC) restricted recognition pathway. Inhibition assays showed the mechanism of cytotoxicity to be calcium-dependent, granule exocytosis pathway, rather than the internal disintegration pathway. Cold target competition assays indicated that a common CTL population contributed to the recognition of autologous and allogeneic-infected targets. These effectors showed recognition of infected targets which was distinct from that of K562 cells. Non-MHC restricted lysis-associated molecule 2B4 (CD244) was upregulated on culturing and made a significant contribution to lysis of FcgammaR-bearing targets in a redirected killing assay. These findings suggest that CTL can recognize virally infected cells through a combination of non-MHC restricted mechanisms and may result in more efficient lysis than classical CD8+ T cells.

Equivalent helper functions of human “naive” and “memory” CD4+ T cells for the generation of alloreactive cytotoxic T lymphocytes

Cells comprising the CD4+ T-cell population are heterogeneous with regard to function, maturation, and the expression of membrane molecules such as the CD45RA antigen. Previous analyses of the CD4+ subsets defined by CD45RA antigen expression have shown that the ability to provide help for antibody production is restricted to cells within the CD4+CD45RA- subset. In the present studies, we have examined the ability of "naive" CD4+CD45RA+ cells and "memory" CD4+CD45RA- cells to provide help for the generation of alloreactive CD8+ cytotoxic T lymphocytes. When purified CD4+CD45RA+ or CD4+CD45RA- cells were cultured with autologous CD8+ cells and allogeneic E- stimulator cells, both subsets were consistently able to provide help for CD8+ cytotoxic T-cell development. In contrast, the ability to provide help for antibody production was restricted to cells in the CD4+CD45RA- subset. Differences in the mechanisms of the helper functions for these two systems were also identified. Whereas exogenous interleukin-2 (IL-2) could replace the help provided by either CD4+ subset for cytotoxic T-cell generation, IL-2 had only minimal effects on immunoglobulin production. Thus, our studies highlight the contrasting cellular requirements and mechanisms involved in "help" for B-cell differentiation versus cytotoxic T-lymphocyte generation, and they show that the helper/inducer functions of human CD4+ cells are not mediated solely by the CD4+CD45RA- subset.