Functional study of CD4+CD25+ regulatory T cells in health and autoimmune hepatitis

Regulatory CD4(+)CD25(+) T cells (Tregs) are defective numerically and functionally in autoimmune hepatitis (AIH). We have investigated and compared the mechanism of action of Tregs in healthy subjects and in AIH patients using Transwell experiments, where Tregs are cultured either in direct contact with or separated from their targets by a semipermeable membrane. We also studied Treg FOXP3 expression and effect on apoptosis. Direct contact is necessary for Tregs to suppress proliferation and IFN-gamma production by CD4(+)CD25(-) and CD8(+) T cells in patients and controls. Moreover, in both, direct contact of Tregs with their targets leads to increased secretion of regulatory cytokines IL-4, IL-10, and TGF-beta, suggesting a mechanism of linked immunosuppression. Tregs/CD4(+)CD25(-) T cell cocultures lead to similar changes in IFN-gamma and IL-10 secretion in patients and controls, whereas increased TGF-beta secretion is significantly lower in patients. In contrast, in patients, Tregs/CD8(+) T cell cocultures lead to a higher increase of IL-4 secretion. In AIH, Treg FOXP3 expression is lower than in normal subjects. Both in patients and controls, FOXP3 expression is present also in CD4(+)CD25(-) T cells, although at a low level and not associated to suppressive function. Both in patients and controls, addition of Tregs does not influence target cell apoptosis, but in AIH, spontaneous apoptosis of CD4(+)CD25(-) T cells is reduced. In conclusion, Tregs act through a direct contact with their targets by modifying the cytokine profile and not inducing apoptosis. Deficient CD4(+)CD25(-) T cell spontaneous apoptosis may contribute to the development of autoimmunity.     

Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy

JC virus (JCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML) and cross-recognize the polyomavirus BK virus (BKV). We sought to determine the frequency and phenotype in fresh blood of CD8+ T cells specific for two A*0201-restricted JCV epitopes, VP1(p36) and VP1(p100), and assess their impact on JC and BK viremia and viruria in 15 healthy subjects, eight human immunodeficiency virus-positive (HIV+) individuals, and nine HIV+ patients with PML (HIV+ PML patients) classified as survivors. After magnetic pre-enrichment of CD8+ T cells, epitope-specific cells ranged from 0.001% to 0.022% [corrected] by tetramer staining, with no significant difference among the three study groups. By use of seven-color flow cytometry, there was no predominant differentiation phenotype subset among JCV-specific CD8+ T cells in healthy individuals, HIV+ subjects, or HIV+ PML patients. However, in one HIV+ PML patient studied in the acute phase, there was a majority of activated effector memory cells. BKV DNA was undetectable in all blood samples by quantitative PCR, while a low JC viral load was found in the blood of only one HIV+ and two HIV+ PML patients. JCV and BKV DNA were detected in 33.3% and 13.3% of all urine samples, respectively, independent of the presence of JCV-specific CTL. The detection of JCV DNA in the urine was associated with the presence of a JCV VP1(p100) CTL response. Immunotherapies aiming at increasing the cellular immune response against JCV may be valuable in the treatment of HIV+ individuals with PML.     

Role of CD4+ and CD8+ T-cell responses against JC virus in the outcome of patients with progressive multifocal leukoencephalopathy (PML) and PML with immune reconstitution inflammatory syndrome

Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the brain caused by JC virus (JCV). To assess the role of CD4(+) and CD8(+) T-cells against JCV in the clinical outcome of PML and PML in the setting of immune reconstitution inflammatory syndrome (IRIS), we tested gamma interferon (IFN-γ) response by enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining (ICS) in 117 subjects, including 66 PML patients with different clinical outcomes. Both assays were concordant and demonstrated that the cellular immune response against JCV is associated with better clinical outcome. PML survivors had an early CD8(+) T-cell response more frequently than PML progressors (100% versus 27.3%; P = 0.001), while only a trend was observed for the early CD4(+) T-cell response between these two groups (80% versus 45.5%; P = 0.18). Although IRIS itself was more frequent in the PML survivor group, there was no difference in IFN-γ-producing CD4(+) and CD8(+) T-cells between IRIS and non-IRIS PML patients, suggesting that T-cells expressing other cytokines likely have a role in the immunopathogenesis of IRIS. ELISpot and ICS assays are useful prognostic markers of PML evolution and may help in the clinical management of these patients.     

Cytologic findings in progressive multifocal leukoencephalopathy. Report of two cases

Cytologic preparations from two cases of progressive multifocal leukoencephalopathy (PML) were obtained by stereotactically guided needle biopsies using computerized tomograms of the brain. Case 1 was a 32-year-old man with an acquired immunodeficiency syndrome-related complex. Case 2 was a 71-year-old man with chronic lymphocytic leukemia. Smears showed moderate cellularity, consisting of moderately to markedly atypical cells with enlarged hyperchromatic nuclei. The chromatin pattern showed smudging, with or without clumping, similar in pattern to the human polyomavirus-infected "decoy" cells seen in urine cytology. Nuclei were predominantly round to oval, smoothly contoured and often stripped of cytoplasm. Occasional bizarre lobulated or multinucleated forms were seen. Some atypical cells had abundant cytoplasm exhibiting stellate projections. Histologic sections of the biopsy material confirmed the diagnosis in each case. In both cases, electron microscopy demonstrated intranuclear polyoma-type virus particles. The present findings suggest that PML should be considered in the differential diagnosis of marked cytologic atypia in brain aspirates from immunocompromised patients.     

JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the polyomavirus JC (JCV) within a setting of immunosuppression. The nature of the immune response that contains replication of this virus is unknown. We have explored JCV-specific cellular immune responses in patients with PML and control subjects. JCV antigen-stimulated peripheral blood mononuclear cells (PBMC) of four human immunodeficiency virus (HIV)-infected patients who were survivors of PML and one HIV-uninfected patient recently diagnosed with PML lysed autologous B-lymphoblastoid cell lines expressing either the JCV T regulatory protein or the VP1 major capsid protein. This lysis was mediated by CD8(+) T lymphocytes and was major histocompatibility complex class I restricted. These cells were therefore cytotoxic T lymphocytes (CTL). JCV-specific CTL could not be detected in PBMC of three HIV-infected PML patients who had progressive neurologic disease and an eventual fatal outcome. These data suggest that the JCV-specific cellular immune response may play a crucial role in the containment of PML. This finding may also prove useful as a favorable prognostic marker in the clinical management of these patients.     

Functional heterogeneity among CD4+ encephalitogenic T cells in recruitment of CD8+ T cells in experimental autoimmune encephalomyelitis

Inoculation of Lewis rats with live or attenuated (irradiated or paraformaldehyde-fixed) CD4+ encephalitogenic T cells (S1 line) protects the recipients from transferred experimental autoimmune encephalomyelitis (tEAE) induced by S1 cells. A CD8+ T lymphocyte population specifically activated against the EAE-inducing S1 cells can be readily isolated from the lymphoid organs of pretreated animals. We show, in the present study, that encephalitogenic T cell lines derived from Lewis rats differ in their ability to induce resistance against tEAE in vivo and to stimulate CD8+ cell proliferation in vitro. We also demonstrate that the S19 line of encephalitogenic T cells, in combination with myelin basic protein (MBP), can stimulate CD8+ cell proliferation in vitro. The CD8+ cells generated in this way strongly suppress MBP-specific T cell proliferation in vitro. This combined effect of T cells and MBP was also evident in vivo. Neither S19 cells nor MBP alone induced resistance against S19-mediated tEAE, rather coinjection of these cells and MBP was required. Our results suggest that resistance to EAE is mediated by distinct populations of encephalitogenic T cells that activate Ts cells through different mechanisms. In some instances, both autoreactive T cells and their relevant autoantigen(s) may be needed to activate Ts cells in vivo.     

Neurotoxicity that may mimic progressive multifocal leukoencephalopathy in patient with transplanted kidney

We present the 55-year-old woman who has had kidney transplantation three times. She has been treated with immunosuppressive therapy and lamivudine for hepatitis B and C. Nine years after the last transplantation she showed neurological symptoms that presented in the form of confusion and epileptic seizures of the grand mal type. A brain MRI showed large oval zones of hyperintense MR signal in T2-weighted image and hypointense in T1-weighted image around the frontal horns of the lateral ventricles, bilaterally and in both cerebellar hemispheres. After reduction in immunosuppression and the exclusion of lamivudine from therapy, the patient was stable with normal neurological status during the course of next five years. We start from the assumption that the concomitant use of cyclosporin with mycophenolate mofetil and lamivudine, despite normal concentrations of cyclosporin, might cause the accumulation of toxic metabolites and lead to neurotoxicity that mimics PML in a chronic viral environment.     

Autophagy in CD4+ T-cell immunity and tolerance

Autophagy is a homeostatic process that enables eukaryotic cells to deliver cytoplasmic constituents for lysosomal degradation, to recycle nutrients and to survive during starvation. In addition to these primordial functions, autophagy has emerged as a key mechanism in orchestrating innate and adaptive immune responses to intracellular pathogens. Autophagy restricts viral infections as well as replication of intracellular bacteria and parasites and delivers pathogenic determinants for TLR stimulation and for MHC class II presentation to the adaptive immune system. Apart from its role in defense against pathogens, autophagy-mediated presentation of self-antigens in the steady state could have a crucial role in the induction and maintenance of CD4(+) T-cell tolerance. This review describes the mechanisms by which the immune system utilizes autophagic degradation of cytoplasmic material to regulate adaptive immune responses.     

Suppression in murine experimental autoimmune thyroiditis: in vivo inhibition of CD4+ T cell-mediated resistance by a nondepleting rat CD4 monoclonal antibody.

Genetically susceptible mice become resistant to experimental autoimmune thyroiditis (EAT) induction with mouse thyroglobulin (MTg) and lipopolysaccharide after pretreatment with deaggregated MTg (dMTg). Recent work showed this suppression to be mediated by CD4+ suppressor T cells (Ts). To study Ts action in vivo, we used a rat IgG2a monoclonal antibody (mAb), YTS 177.9, which modulates CD4 antigen in vivo without depleting CD4+ cells. Initial studies showed that after two 1-mg doses of mAb 7 days apart, extensive CD4 antigen modulation of peripheral blood leukocytes occurred within 4 days. Mice given CD4 mAb 24 hr before dMTg (2 doses, 7 days apart) were resistant to EAT induction when immunized with MTg and LPS 20 days later. Also, anti-rat IgG2a titers were reduced following challenge with heat-aggregated rat IgG2a compared to controls. Subsequent analysis of serum in CD4 mAb-treated animals revealed that mAb was present in the circulation for 14 days. Moreover, mice given CD4 mAb and dMTg, then challenged after only 10 days, when CD4 mAb was still circulating, developed a significantly higher incidence of thyroid damage than controls. These findings suggest that modulation of CD4 antigen does not interfere with Ts activation, but the presence of CD4 mAb, at the time of autoantigenic challenge, can interfere with tolerance to EAT induction. Thus, the direct relationship between the presence of CD4 mAb and inhibition of EAT suppression implicates a role for CD4 molecules in the mediation of suppression.     

Analysis of JC virus DNA purified directly from human progressive multifocal leukoencephalopathy brains.

Human polyomavirus JC DNA was purified directly from the diseased brain tissue of two patients with progressive multifocal leukoencephalopathy (PML) by a method employing differential salt precipitation (B. Hirt, J. Mol. Biol. 26:365-369, 1967). Each of the viral genomes (JC-NIH-1 and JC-NIH-2) was molecularly cloned intact in Escherichia coli, using pBR322, at their unique EcoRI (0.00 map unit) and BamHI (0.51 map unit) sites. The JC-NIH-1 genome was approximately 50 base pairs larger and the JC-NIH-2 genome was approximately 50 base pairs smaller than the prototype human polyomavirus JC (Mad-1) DNA. Analysis of the restriction endonuclease cleavage fragments of these two DNAs and the human polyomavirus JC (Mad-1) DNA revealed only slight differences which mapped in a region of the genome extending from 0.67 to 0.74 map unit. From previous homology studies, this region of variance corresponds to the noncoding region to the late side of the origin of DNA replication.     

The role of CD4+CD25+ immunoregulatory T cells in the induction of autoimmune gastritis

A number of experimental models of organ-specific autoimmunity involve a period of peripheral lymphopenia prior to disease onset. There is now considerable evidence that the development of autoimmune disease in these models is due to the absence of CD4+CD25+ regulatory T cells. However, the role of CD4+CD25+ regulatory T cells in the prevention of autoimmune disease in normal individuals has not been defined. Here we have assessed the affect of depletion of CD4+CD25+ regulatory T cells in BALB/c mice on the induction of autoimmune gastritis. The CD4+CD25+ T cell population was reduced to 95% of the original population in adult thymectomized mice by treatment with anti-CD25 mAb. By 48 days after the anti-CD25 treatment, the CD4+CD25+ regulatory T cell population had returned to a normal level. Treatment of thymectomized adult mice for up to 4 weeks with anti-CD25 mAb did not result in the development of autoimmune gastritis. Furthermore, we have demonstrated that depletion of CD4+CD25+ regulatory T cells, together with transient CD4+ T lymphopenia, also did not result in the development of autoimmune gastritis, indicating that peripheral expansion of the CD4+ T cell population, per se, does not result in autoimmunity in adult mice. On the other hand, depletion of CD4+CD25+ T cells in 10-day-old euthymic mice resulted in a 30% incidence of autoimmune gastritis. These data suggest that CD4+CD25+ regulatory T cells may be important in protection against autoimmunity while the immune system is being established in young animals, but subsequently other factors are required to initiate autoimmunity.     

CD11b+ monocytes abrogate Th17 CD4+ T cell-mediated experimental autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) represents a Th17 T cell-mediated mouse model of postinflammatory heart disease. In BALB/c wild-type mice, EAM is a self-limiting disease, peaking 21 days after alpha-myosin H chain peptide (MyHC-alpha)/CFA immunization and largely resolving thereafter. In IFN-gammaR(-/-) mice, however, EAM is exacerbated and shows a chronic progressive disease course. We found that this progressive disease course paralleled persistently elevated IL-17 release from T cells infiltrating the hearts of IFN-gammaR(-/-) mice 30 days after immunization. In fact, IL-17 promoted the recruitment of CD11b(+) monocytes, the major heart-infiltrating cells in EAM. In turn, CD11b(+) monocytes suppressed MyHC-alpha-specific Th17 T cell responses IFN-gamma-dependently in vitro. In vivo, injection of IFN-gammaR(+/+)CD11b(+), but not IFN-gammaR(-/-)CD11b(+), monocytes, suppressed MyHC-alpha-specific T cells, and abrogated the progressive disease course in IFN-gammaR(-/-) mice. Finally, coinjection of MyHC-alpha-specific, but not OVA-transgenic, IFN-gamma-releasing CD4(+) Th1 T cell lines, together with MyHC-alpha-specific Th17 T cells protected RAG2(-/-) mice from EAM. In conclusion, CD11b(+) monocytes play a dual role in EAM: as a major cellular substrate of IL-17-induced inflammation and as mediators of an IFN-gamma-dependent negative feedback loop confining disease progression.     

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.     

CTLA-4 and CD4+ CD25+ regulatory T cells inhibit protective immunity to filarial parasites in vivo

The T cell coinhibitory receptor CTLA-4 has been implicated in the down-regulation of T cell function that is a quintessential feature of chronic human filarial infections. In a laboratory model of filariasis, Litomosoides sigmodontis infection of susceptible BALB/c mice, we have previously shown that susceptibility is linked both to a CD4+ CD25+ regulatory T (Treg) cell response, and to the development of hyporesponsive CD4+ T cells at the infection site, the pleural cavity. We now provide evidence that L. sigmodontis infection drives the proliferation and activation of CD4+ Foxp3+ Treg cells in vivo, demonstrated by increased uptake of BrdU and increased expression of CTLA-4, Foxp3, GITR, and CD25 compared with naive controls. The greatest increases in CTLA-4 expression were, however, seen in the CD4+ Foxp3- effector T cell population which contained 78% of all CD4+ CTLA-4+ cells in the pleural cavity. Depletion of CD25+ cells from the pleural CD4+ T cell population did not increase their Ag-specific proliferative response in vitro, suggesting that their hyporesponsive phenotype is not directly mediated by CD4+ CD25+ Treg cells. Once infection had established, killing of adult parasites could be enhanced by neutralization of CTLA-4 in vivo, but only if performed in combination with the depletion of CD25+ Treg cells. This work suggests that during filarial infection CTLA-4 coinhibition and CD4+ CD25+ Treg cells form complementary components of immune regulation that inhibit protective immunity in vivo.     

The relative importance of CD4+ and CD8+T cells in immunity to pulmonary paracoccidioidomycosis

Protective immunity in paracoccidioidomycosis (PCM) is believed to be mediated by cellular immunity, but the role of T cell subsets has never been investigated. The aim of this study was to characterize the function of CD4+ and CD8+ T cells in the immunity developed by susceptible, intermediate and resistant mice after P. brasiliensis infection. In susceptible mice, depletion of CD4+ T cells did not alter disease severity and anergy of cellular immunity but diminished antibody production. Anti-CD8 treatment led to increased fungal loads, but restored DTH reactivity. In resistant mice, both CD4+ and CD8+ T cells control fungal burdens and cytokines although only the former regulate DTH reactions and antibody production. In the intermediate strain, deficiency of whole T and CD8+ T cells but not of CD4+ T or B cells led to increased mortality rates. Thus, in pulmonary PCM: (a) irrespective of the host susceptibility pattern, fungal loads are mainly controlled by CD8+ T cells, whereas antibody production and DTH reactions are regulated by CD4+ T cells; (c) CD4+ T cells play a protective role in the resistant and intermediate mouse strains, whereas in susceptible mice they are deleted or anergic; (d) genetic resistance to PCM is associated with concomitant CD4+ and CD8+ T cell immunity secreting type 1 and type 2 cytokines.     

Infections that induce autoimmune diabetes in BBDR rats modulate CD4+CD25+ T cell populations

Viruses are believed to contribute to the pathogenesis of autoimmune type 1A diabetes in humans. This pathogenic process can be modeled in the BBDR rat, which develops pancreatic insulitis and type 1A-like diabetes after infection with Kilham's rat virus (RV). The mechanism is unknown, but does not involve infection of the pancreatic islets. We first documented that RV infection of BBDR rats induces diabetes, whereas infection with its close homologue H-1 does not. Both viruses induced similar humoral and cellular immune responses in the host, but only RV also caused a decrease in splenic CD4(+)CD25(+) T cells in both BBDR rats and normal WF rats. Surprisingly, RV infection increased CD4(+)CD25(+) T cells in pancreatic lymph nodes of BBDR but not WF rats. This increase appeared to be due to the accumulation of nonproliferating CD4(+)CD25(+) T cells. The results imply that the reduction in splenic CD4(+)CD25(+) cells observed in RV-infected animals is virus specific, whereas the increase in pancreatic lymph node CD4(+)CD25(+) cells is both virus and rat strain specific. The data suggest that RV but not H-1 infection alters T cell regulation in BBDR rats and permits the expression of autoimmune diabetes. More generally, the results suggest a mechanism that could link an underlying genetic predisposition to environmental perturbation and transform a "regulated predisposition" into autoimmune diabetes, namely, failure to maintain regulatory CD4(+)CD25(+) T cell function.     

Escape of malaria parasites from host immunity requires CD4+ CD25+ regulatory T cells

Infection with malaria parasites frequently induces total immune suppression, which makes it difficult for the host to maintain long-lasting immunity. Here we show that depletion of CD4(+)CD25(+) regulatory T cells (T(reg)) protects mice from death when infected with a lethal strain of Plasmodium yoelii, and that this protection is associated with an increased T-cell responsiveness against parasite-derived antigens. These results suggest that activation of T(reg) cells contributes to immune suppression during malaria infection, and helps malaria parasites to escape from host immune responses.     

Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory

Emerging evidence indicates that CD8+ and CD4+ T-cell immunity is differentially regulated. Here we have delineated differences and commonalities among antiviral T-cell responses by enumeration and functional profiling of eight specific CD8+ and CD4+ T-cell populations during primary, memory and recall responses. A high degree of coordinate regulation among all specific T-cell populations stood out against an approximately 20-fold lower peak expansion and prolonged contraction phase of specific CD4+ T-cell populations. Surprisingly, although CD8+ T-cell memory was stably maintained for life, levels of specific CD4+ memory T cells gradually declined. However, this decay, which seemed to result from less efficient rescue from apoptosis, did not affect functionality of surviving virus-specific CD4+ T cells. Our results indicate that CD4+ T-cell memory might become limiting under physiological conditions and that conditions precipitating CD4+ T-cell loss might compromise protective immunity even in the presence of unimpaired CD8+ T-cell responses.     

Protective cellular retroviral immunity requires both CD4+ and CD8+ immune T cells.

We have found previously that postexposure chemoprophylaxis with 3'-azido-3'-deoxythymidine (also known as zidovudine or AZT) in combination with recombinant human alpha A/D interferon fully protected mice exposed to a lethal dose of Rauscher murine leukemia virus (RLV) against viremia and disease. After cessation of therapy, over 90% of these mice were able to resist rechallenge with live RLV, thus demonstrating an acquired immunity. Adoptive cell transfer of 4 x 10(7) cells from immunized mice fully protected naive recipients from viremia and splenomegaly after RLV challenge. However, when these immune T cells were fractionated into CD4+ and CD8+ subpopulations, only partial protection was found when 4 x 10(7) T cells of either subset were given. Full protection against RLV challenge was seen again when the T-cell subsets from immunized mice were recombined and transferred at the same number into naive mice. We conclude that cellular immunity alone is protective and that both CD4+ and CD8+ cell types are required for conferring full protection against live virus challenge.